U.S. patent application number 17/528173 was filed with the patent office on 2022-05-19 for methods for regressing or reversing fibrosis and/or liver cirrhosis in a subject in need thereof using high-dose niacin, or a niacin analog thereof.
The applicant listed for this patent is The Regents of the University of California, United States Government As Represented By The Department of Veterans Affairs. Invention is credited to Shobha Kamanna, Vaijinath Kamanna, Moti Lal Kashyap.
Application Number | 20220152012 17/528173 |
Document ID | / |
Family ID | |
Filed Date | 2022-05-19 |
United States Patent
Application |
20220152012 |
Kind Code |
A1 |
Kashyap; Moti Lal ; et
al. |
May 19, 2022 |
METHODS FOR REGRESSING OR REVERSING FIBROSIS AND/OR LIVER CIRRHOSIS
IN A SUBJECT IN NEED THEREOF USING HIGH-DOSE NIACIN, OR A NIACIN
ANALOG THEREOF
Abstract
The disclosure provides methods to reverse or regress fibrosis
and/or liver cirrhosis in a subject in need thereof, using
high-dose (pharmacologic) niacin, or a niacin analog thereof.
Inventors: |
Kashyap; Moti Lal; (Rancho
Palos Verdes, CA) ; Kamanna; Vaijinath; (Irvine,
CA) ; Kamanna; Shobha; (Irvine, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California
United States Government As Represented By The Department of
Veterans Affairs |
Oakland
Washington |
CA
DC |
US
US |
|
|
Appl. No.: |
17/528173 |
Filed: |
November 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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63114494 |
Nov 16, 2020 |
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International
Class: |
A61K 31/455 20060101
A61K031/455; A61P 1/16 20060101 A61P001/16; A61K 45/06 20060101
A61K045/06 |
Claims
1. A method to reverse or regress fibrosis and/or liver cirrhosis
in a subject in need thereof, comprising: administering to a
subject having fibrosis and/or liver cirrhosis one or more
pharmaceutical doses of a pharmaceutical composition comprising
niacin, or of a niacin analog thereof, wherein the pharmaceutical
composition comprises 250 mg to 2000 mg of niacin, or niacin
equivalent dosing of a niacin analog thereof, wherein the subject
is administered a total daily dose of 250 mg to 6000 mg of niacin,
or niacin equivalent dosing of a niacin analog thereof, and wherein
administration of the one or more pharmaceutical doses of niacin or
a niacin analog thereof reverses or regresses fibrosis and/or liver
cirrhosis in the subject.
2. The method of claim 1, wherein the fibrosis is associated with
elevated or overaccumulation of collagen in cells or tissue.
3. The method of claim 2, wherein administration of one or more
pharmaceutical doses of niacin or of a niacin analog to the subject
reduces collagen levels in fibrotic tissue.
4. The method of claim 1, wherein administration of one or more
pharmaceutical doses of niacin or of a niacin analog stabilizes or
normalizes the expression levels of matrix metalloproteinases
(MMPs) and/or tissue inhibitors of metalloproteinases (TIMPs).
5. The method of claim 1, wherein the fibrosis affects one or more
tissues or organs.
6. The method of claim 5, wherein the one or more tissues or organs
are selected from liver, bone marrow, lung, kidney,
gastrointestinal tract, skin, eye, endomyocardium, musculoskeletal
system, and myocardium.
7. The method of claim 6, wherein the one or more tissues or organs
is the liver.
8. The method of claim 1, wherein the subject has a disease,
disorder, or condition selected from the group consisting of a
cystic fibrosis, idiopathic pulmonary fibrosis, post COVID-19
fibrosis, radiation-induced lung injury, liver fibrosis, liver
cirrhosis, glial scars, arterial stiffness, arthrofibrosis, Crohn's
disease, Dupuytren's contracture, keloids, mediastinal fibrosis,
myelofibrosis, Peyronie's disease, nephrogenic system fibrosis,
progressive massive fibrosis, retroperitoneal fibrosis,
scleroderma/systemic sclerosis, adhesive capsulitis, interstitial
fibrosis, replacement fibrosis, inflammatory bowel disease, renal
fibrosis in patients with tubulointerstitial fibrosis,
glomerulosclerosis, lung fibrosis, and chronic kidney disease.
9. The method of claim 1, wherein the subject has grade 1, grade 2,
grade 3, or grade 4 liver fibrosis.
10. The method of claim 1, wherein the subject has liver
cirrhosis.
11. The method of claim 1, wherein the subject has liver fibrosis
resulting from a biliary obstruction, iron overload, autoimmune
hepatitis, Wilson's disease, a viral hepatitis B infection, or a
viral hepatitis C infection.
12. The method of claim 1, wherein the niacin analog is selected
from nicotinamide, 6-hydroxy nicotinamide, N-methyl-nicotinamide,
acifran, acipimox, niceritrol, ARI-3037MO, and nicotinamide
riboside chloride.
13. The method of claim 1, wherein the pharmaceutical composition
is formulated for oral, transdermal or parenteral delivery.
14. The method of claim 13, wherein the pharmaceutical composition
is formulated as an extended-release or time-release formulation
for oral delivery.
15. The method of claim 14, wherein the pharmaceutical composition
is formulated as a film-coated extended-release tablet.
16. The method of claim 15, wherein the film-coated
extended-release tablet comprises hypromellose, povidone, stearic
acid, polyethylene glycol, and/or coloring reagents.
17. The method of claim 13, wherein the pharmaceutical composition
is formulated as a tablet and comprises croscarmellose sodium,
hydrogenated vegetable oil, magnesium stearate and/or
microcrystalline cellulose.
18. The method of claim 1, wherein the one or more pharmaceutical
doses are administered sequentially or concurrently with one or
more therapeutics selected from anti-fibrotic therapeutics,
prostaglandin D2 binding drugs, antivirals, gallstone solubilizing
agents, anti-thrombotic treatments, nonalcoholic fatty liver
disease (NAFLD) treatments, nonalcoholic steatohepatitis (NASH)
treatments, sepsis treatments, anti-mycobacterial agents, chelation
therapy agents, anti-bacterial agents, anti-fungal agents,
steroidal drugs, anticoagulants, non-steroidal anti-inflammatory
agents, antiplatelet agents, norepinephrine reuptake inhibitors
(NRIs), dopamine reuptake inhibitors (DRIs), Serotonin and
norepinephrine reuptake inhibitors (SNRIs), sedatives,
Norepinephrine and Dopamine Reuptake Inhibitors (NDRIs),
serotonin-norepinephrine-dopamine reuptake inhibitors (SNDRIs),
monoamine oxidase inhibitors, hypothalamic phospholipids,
Endothelin converting enzymes (ECE) inhibitors, opioids,
thromboxane receptor antagonists, potassium channel openers,
thrombin inhibitors, hypothalamic phospholipids, growth factor
inhibitors, anti-platelet agents, P2Y(AC) antagonists,
anticoagulants, low molecular weight heparins, Factor VIa
Inhibitors and Factor Xa Inhibitors, renin inhibitors, neutral
endopeptidase (NEP) inhibitors, vasopepsidase inhibitors, HMG CoA
reductase inhibitors, squalene synthetase inhibitors, fibrates,
bile acid sequestrants, anti-atherosclerotic agents, microsomal
triglyceride transfer protein (MTP) Inhibitors, calcium channel
blockers, potassium channel activators, alpha-muscarinic agents,
beta-muscarinic agents, antiarrhythmic agents, diuretics,
thrombolytic agents, anti-diabetic agents, mineralocorticoid
receptor antagonists, growth hormone secretagogues, aP2 inhibitors,
phosphodiesterase inhibitors, protein tyrosine kinase inhibitors,
anti-inflammatories, anti-proliferatives, chemotherapeutic agents,
immunosuppressants, anticancer agents and cytotoxic agents,
anti-metabolites, antibiotics, farnesyl-protein transferase
inhibitors, hormonal agents, microtubule-disruptor agents,
microtubule-stabilizing agents, plant-derived products,
epipodophyllotoxins, taxanes, topoisomerase inhibitors,
prenyl-protein transferase inhibitors, cyclosporins, cytotoxic
drugs, tumor necrosis factor (TNF)-alpha inhibitors, anti-TNF
antibodies and soluble TNF receptors, cyclooxygenase-2 (COX-2)
inhibitors, galectin inhibitors, transforming growth factor
(TGF)-beta inhibitors, anti-TGF-beta antibodies, anti-oxidants,
oxidative stress inhibitors, TIMP inhibitors, matrix
metalloproteinase-1 (MMP) activators, kynurenic acid, FS2,
cenicriviroc, aramchol, aramchol meglumine, and belapectin.
19. The method of claim 18, wherein the one or more pharmaceutical
doses are administered sequentially or concurrently with one or
more anti-fibrotic therapeutics.
20. The method of claim 1, wherein the one or more pharmaceutical
doses are administered sequentially or concurrently with one or
more therapeutics selected from laropiprant, cenicriviroc,
resmetirom, ocaliva, elafibranor, aramchol, IMM124E, semaglutide,
lanifibranor, seladelpar, belapectin, PXL_065, MADC_0602,
aldafermin, VK2809, EDP_305, PF_05221304, tipelukast, tropifexor,
DF102, LMB763, nitazoxanide, tesamorelin, TERN_101, lazarotide,
BMS986036, Saroglitazar, AKR001, CRV431, GRI_0621, EYP001,
BMS_986171, isosabutate, PF_06835919, PF_06865571, nalmefene,
LIK066, BIO89_100, Namodenoson, MT_3995, pemafibrate, PXL770,
gemcabene, foralumab, SGM_1019, KBP_042, hepastem, CER_209, DUR928,
sotagliflozin, elobixibat, SAR425899, NGM313, namacizumab,
TERN_201, LPCN_1144, ND_L02_S0201, RTU_1096, IONIS_DGAT2R,
bezafibrate, INT_767, NP160, NEULIV, NP135, BFKB8488A, NC_001,
VK0214, HM15211, CM_101, AZD2693, NV556, SP_1373, RLBN1127,
RYI_018, NVP022, VPR_423, CB4209-CB4211, and GKT_137831.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn. 119
from Provisional Application Ser. No. 63/114,494, filed Nov. 16,
2020, the disclosure of which is incorporated herein by
reference.
TECHNICAL FIELD
[0002] The disclosure provides methods to reverse or regress
fibrosis and/or liver cirrhosis in a subject in need thereof, using
high-dose (pharmacologic) niacin, or a niacin analog thereof.
BACKGROUND
[0003] Hepatic fibrosis, a wound healing response of the liver, is
mainly caused by chronic liver injuries with diverse etiologies
including NASH, viral hepatitis, alcoholism and autoimmune liver
diseases. The hallmark of hepatic fibrosis is the excessive
accumulation of extracellular matrix proteins (e.g., collagen type
1) produced by activated hepatic stellate cells. Stellate cells are
quiescent in normal liver, but upon activation by liver injury
these activated hepatic stellate cells are the primary cell types
producing extracellular matrix proteins, leading to hepatic
fibrosis. Increased hepatic oxidative stress and generation of
reactive oxygen species (ROS, an index of oxidative stress) play a
crucial role in stellate cell activation and hepatic fibrosis.
Hepatic fibrosis with persistent deposition of collagen results in
distortion of hepatic parenchyma and vascular structure, clinically
manifesting as liver cirrhosis.
SUMMARY
[0004] Collagen deposition is a major underlying histologic feature
in hepatic fibrosis and manifests clinically as liver cirrhosis and
its complications. Niacin (not as vitamin but as drug) is an
anti-dyslipidemic drug used for treatment of atherosclerotic
cardiovascular disease. Niacin's action on human hepatic fibrosis
is unknown. The efficacy of niacin on regression of preexisting
collagen content and changes in oxidative stress were studied
herein in cultured primary human hepatic stellate cells. The cells
were selected from fresh livers of recently deceased patients with
histologic fibrosis and associated steatosis and inflammation, and
in subjects without fibrosis. Collagen content in stellate cells
from patients were 4-fold higher than in cells from non-fibrosis
subjects. Treatment of stellate cells with pharmacologically
relevant concentrations of niacin (i.e., high-dose niacin) produced
a significant dose and time dependent decrease in pre-existing
collagen by 48-60% at 48 h incubation and 54-65% at 96 h
incubation. In stellate cells from non-fibrosis subjects, niacin
prevented, and suppressed collagen formation induced by oxidative
stressors TGF-.beta. and hydrogen peroxide. Pharmacologic doses of
niacin significantly inhibited production of reactive oxygen
species induced by oxidative stressors: palmitic acid or hydrogen
peroxide, by 52% and 50% respectively. Accordingly, the data
presented herein demonstrated that pharmacologic doses of niacin
regressed or reversed preexisting fibrosis in primary human
stellate cells likely via oxidative stress reduction. As liver
fibrosis manifests clinically as cirrhosis, pharmacologic doses of
niacin, or a niacin analog thereof, can be used for the treatment
of cirrhosis of the liver.
[0005] In a particular embodiment, the disclosure provides a method
to reverse or regress fibrosis and/or liver cirrhosis in a subject
in need thereof, comprising: administering to a subject having
fibrosis and/or liver cirrhosis one or more pharmaceutical doses of
a pharmaceutical composition comprising niacin or of a niacin
analog thereof, wherein the pharmaceutical composition comprise 250
mg to 2000 mg of niacin, or niacin equivalent dosing of a niacin
analog thereof, wherein the subject is administered a total daily
dose of 250 mg to 6000 mg of niacin, or niacin equivalent dosing of
a niacin analog thereof, and wherein administration of the one or
more pharmaceutical doses of niacin or a niacin analog thereof
reverses or regresses fibrosis and/or liver cirrhosis in the
subject. In a further embodiment, the fibrosis is associated with
elevated or overaccumulation of collagen in cells or tissue. In
another or a further embodiment herein, administration of one or
more pharmaceutical doses of niacin or of a niacin analog to the
subject reduces collagen levels in fibrotic tissue. In another or a
further embodiment herein, administration of one or more
pharmaceutical doses of niacin or of a niacin analog stabilizes or
normalizes the expression levels of matrix metalloproteinases
(MMPs) and/or tissue inhibitors of metalloproteinases (TIMPs). In
another or a further embodiment herein, the fibrosis affects one or
more tissues or organs. In another or a further embodiment herein,
the one or more tissues or organs are selected from liver, bone
marrow, lung, kidney, gastrointestinal tract, skin, eye,
endomyocardium, musculoskeletal system, and myocardium. In another
or a further embodiment herein, the one or more tissues or organs
is the liver. In another or a further embodiment herein, the
subject has a disease, disorder, or condition selected from the
group consisting of a cystic fibrosis, idiopathic pulmonary
fibrosis, post COVID-19 fibrosis, radiation-induced lung injury,
liver fibrosis, liver cirrhosis, glial scars, arterial stiffness,
arthrofibrosis, Crohn's disease, Dupuytren's contracture, keloids,
mediastinal fibrosis, myelofibrosis, Peyronie's disease,
nephrogenic system fibrosis, progressive massive fibrosis,
retroperitoneal fibrosis, scleroderma/systemic sclerosis, adhesive
capsulitis, interstitial fibrosis, replacement fibrosis,
inflammatory bowel disease, renal fibrosis in patients with
tubulointerstitial fibrosis, glomerulosclerosis, lung fibrosis, and
chronic kidney disease. In another or a further embodiment herein,
the subject has grade 1, grade 2, grade 3, or grade 4 liver
fibrosis. In another or a further embodiment herein, the subject
has grade 1, grade 2, or grade 3 liver fibrosis. In another or a
further embodiment herein, the subject has liver cirrhosis. In
another or a further embodiment herein, the subject has liver
fibrosis and nonalcoholic steatohepatitis, or liver fibrosis and
alcoholic steatohepatitis. In another or a further embodiment
herein, the subject has liver fibrosis resulting from a biliary
obstruction, iron overload, autoimmune hepatitis, Wilson's disease,
a viral hepatitis B infection, or a viral hepatitis C infection. In
another or a further embodiment herein, the niacin analog is
selected from nicotinamide, 6-hydroxy nicotinamide,
N-methyl-nicotinamide, acifran, acipimox, niceritrol, ARI-3037MO,
and nicotinamide riboside chloride. In another or a further
embodiment herein, the pharmaceutical composition is formulated for
oral, transdermal or parenteral delivery. In another or a further
embodiment herein, the pharmaceutical composition is formulated as
an extended-release or time-release formulation for oral delivery.
In another or a further embodiment herein, the pharmaceutical
composition is formulated as a film-coated extended-release tablet.
In another or a further embodiment herein, the film-coated
extended-release tablet comprises hypromellose, povidone, stearic
acid, polyethylene glycol, and/or coloring reagents. In another or
a further embodiment herein, the pharmaceutical composition is
formulated as a tablet and comprises croscarmellose sodium,
hydrogenated vegetable oil, magnesium stearate and/or
microcrystalline cellulose. In another or a further embodiment
herein, the one or more pharmaceutical doses are administered
sequentially or concurrently with one or more therapeutics selected
from anti-fibrotic therapeutics, prostaglandin D2 binding drugs,
antivirals, gallstone solubilizing agents, anti-thrombotic
treatments, nonalcoholic fatty liver disease (NAFLD) treatments,
nonalcoholic steatohepatitis (NASH) treatments, sepsis treatments,
anti-mycobacterial agents, chelation therapy agents, anti-bacterial
agents, anti-fungal agents, steroidal drugs, anticoagulants,
non-steroidal anti-inflammatory agents, antiplatelet agents,
norepinephrine reuptake inhibitors (NRIs), dopamine reuptake
inhibitors (DRIs), Serotonin and norepinephrine reuptake inhibitors
(SNRIs), sedatives, Norepinephrine and Dopamine Reuptake Inhibitors
(NDRIs), serotonin-norepinephrine-dopamine reuptake inhibitors
(SNDRIs), monoamine oxidase inhibitors, hypothalamic phospholipids,
Endothelin converting enzymes (ECE) inhibitors, opioids,
thromboxane receptor antagonists, potassium channel openers,
thrombin inhibitors, hypothalamic phospholipids, growth factor
inhibitors, anti-platelet agents, P2Y(AC) antagonists,
anticoagulants, low molecular weight heparins, Factor VIa
Inhibitors and Factor Xa Inhibitors, renin inhibitors, neutral
endopeptidase (NEP) inhibitors, vasopepsidase inhibitors, HMG CoA
reductase inhibitors, squalene synthetase inhibitors, fibrates,
bile acid sequestrants, anti-atherosclerotic agents, microsomal
triglyceride transfer protein (MTP) Inhibitors, calcium channel
blockers, potassium channel activators, alpha-muscarinic agents,
beta-muscarinic agents, antiarrhythmic agents, diuretics,
thrombolytic agents, anti-diabetic agents, mineralocorticoid
receptor antagonists, growth hormone secretagogues, aP2 inhibitors,
phosphodiesterase inhibitors, protein tyrosine kinase inhibitors,
anti-inflammatories, anti-proliferatives, chemotherapeutic agents,
immunosuppressants, anticancer agents and cytotoxic agents,
anti-metabolites, antibiotics, farnesyl-protein transferase
inhibitors, hormonal agents, microtubule-disruptor agents,
microtubule-stabilizing agents, plant-derived products,
epipodophyllotoxins, taxanes, topoisomerase inhibitors,
prenyl-protein transferase inhibitors, cyclosporins, cytotoxic
drugs, tumor necrosis factor (TNF)-alpha inhibitors, anti-TNF
antibodies and soluble TNF receptors, cyclooxygenase-2 (COX-2)
inhibitors, galectin inhibitors, transforming growth factor
(TGF)-beta inhibitors, anti-TGF-beta antibodies, anti-oxidants,
oxidative stress inhibitors, TIMP inhibitors, matrix
metalloproteinase-1 (MMP) activators, kynurenic acid, FS2,
cenicriviroc, aramchol, aramchol meglumine, and belapectin. In
another or a further embodiment herein, the one or more
pharmaceutical doses are administered sequentially or concurrently
with one or more anti-fibrotic therapeutics. In another or a
further embodiment herein, the one or more anti-fibrotic
therapeutics are selected from nintedanib, pirfenidone, rilonacept,
tocilizumab, rituximab, abatacept, lanifibranor, NCT02503644,
NCT03597933, FCX-103, and SAR100842. In another or a further
embodiment herein, the one or more pharmaceutical doses is
administered sequentially or concurrently with a prostaglandin D2
binding drug. In another or a further embodiment herein, the
prostaglandin D2 binding drug is laropiprant. In another or a
further embodiment herein, the one or more pharmaceutical doses are
administered sequentially or concurrently with one or more
therapeutics selected from NASH treatments, NAFLD treatments,
antiviral drugs, and gallstone solubilizing agents. In another or a
further embodiment herein, the NASH treatments and NAFLD treatments
are selected from orlistat, elafibranor, pioglitazone,
saroglitazar, solithromycin, exenatide, liraglutide, sitagliptin,
vildapliptin, aramchol, obeticholic acid, cenicriviroc,
pentoxifylline, emricasan, simtuzumab, galectin-3, atorvastatin,
pravastatin, cerivastatin, lovastatin, mevastatin, pitavastatin,
rosuvastatin, simvastatin, fluvastatin, NGM-282, GS-4997, IMM-124e,
cysteamine, cystamine, and vitamin E. In another or a further
embodiment herein, the one or more pharmaceutical doses are
administered sequentially or concurrently with one or more
therapeutics that target lipid metabolism and insulin resistance;
one or more therapeutics that target lipotoxicity, oxidative
stress, and inflammation; one or more therapeutics that target
fibrosis and cirrhosis; or a combination thereof. In another or a
further embodiment herein, the one or more pharmaceutical doses are
administered sequentially or concurrently with one or more
therapeutics selected from acetyl-CoA carboxylase (ACC) Inhibitors,
fatty acid synthase inhibitors, icosbutate, eicosapentaenoic acid
analogs/derivatives, omega/n-3 fatty acids, thiazolidinediones
(TZDs), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA
reductase) inhibitors, dibrates, peroxisome proliferated-activated
receptors (PPAR)-alpha/beta/gamma/delta agonists/modulators,
fibroblast growth factor (FGF)-19/21 analogs/modulators,
glucagon-like peptide receptors (GLP-1)
analogs/mimetics/modulators, ketohexokinase inhibitors,
mitochondrial pyruvate carrier inhibitor/modulators, sodium-glucose
cotransporters (SGLTs) inhibitors/modulators, adenosine
monophosphate-activated protein kinase (AMPK)
activators/modulators, stearoyl-CoA dehydrogenase (SCD) inhibitors,
diacylglycerol-acyl transferase-2 (DGAT-2) inhibitors, thyroid
hormone receptor-beta (THR-beta) agonists, glucocorticoid
modulators, dipeptidyl peptidase-4 inhibitors (DPP-4), sodium
glucose co-transporter 2 (SGLT2) inhibitors, anti-diabetes 2
agents, PPAR-alpha/beta/gamma/delta agonists/modulators, farnesoid
X receptor (FXR) agonists/modulators, vitamin E, anti-oxidants,
FGF-19/21 modulators, chemokine 2/5 receptor (CCR2/5)
antagonists/Inhibitors, nicotinamide adenine dinucleotide phosphate
oxidase (NADPH oxidase) inhibitors, and angiotensin receptor
antagonists, caspases inhibitors/modulators, apoptosis
inhibitors/modulators, leukotriene/phosphodiesterase/lipoxygenase
antagonists/inhibitors/modulators, galectin-3
antagonists/modulators, apoptosis signal-regulating kinases (ASK)
inhibitors, lysophosphatidic acid receptor 1 (LPA1) antagonists,
and heat shock proteins (HSP47 and other members) inhibitors. In
another or a further embodiment herein, the one or more
pharmaceutical doses are administered sequentially or concurrently
with one or more therapeutics selected from laropiprant,
cenicriviroc, resmetirom, ocaliva, elafibranor, aramchol, IMM124E,
semaglutide, lanifibranor, seladelpar, belapectin, PXL_065,
MADC_0602, aldafermin, VK2809, EDP_305, PF_05221304, tipelukast,
tropifexor, DF102, LMB763, nitazoxanide, tesamorelin, TERN_101,
lazarotide, BMS986036, Saroglitazar, AKR001, CRV431, GRI_0621,
EYP001, BMS_986171, isosabutate, PF_06835919, PF_06865571,
nalmefene, LIK066, BIO89_100, Namodenoson, MT_3995, pemafibrate,
PXL770, gemcabene, foralumab, SGM_1019, KBP_042, hepastem, CER_209,
DUR928, sotagliflozin, elobixibat, SAR425899, NGM313, namacizumab,
TERN_201, LPCN_1144, ND_L02 S0201, RTU_1096, IONIS_DGAT2R,
bezafibrate, INT_767, NP160, NEULIV, NP135, BFKB8488A, NC_001,
VK0214, HM15211, CM_101, AZD2693, NV556, SP_1373, RLBN1127,
RYI_018, NVP022, VPR_423, CB4209-CB4211, and GKT_137831.
DESCRIPTION OF DRAWINGS
[0006] FIG. 1A-B demonstrates that niacin decreased fibrosis in
hepatic stellate cells from human donor subjects with fibrotic NASH
(Donors 3-7): Human hepatic stellate cells from donor subjects with
varying degree of fibrosis and NASH (NAFLD activity scores 2-5, and
fibrosis scores 1-3) were incubated with pharmacologically relevant
concentrations of niacin (0.25 mM and 0.5 mM) for 48 or 96 hours.
Cells were stained with Sirius Red for collagen content, cellular
photographic images, and quantitation. (A) Representative
photographic images of stellate cells (left to right): "Non-NASH"
from donor 1, "NASH" from donor 6 without niacin, with 0.5 mM
niacin at 48, and 96 h incubation. (B) Composite Mean.+-.SE
collagen content data from all 5 NASH-Fibrosis patient (donors 3-7)
showing the ability of niacin to regress pre-existing fibrosis in
stellate cells from patients with NASH and fibrosis. Left bar
marked "Normal" refers to mean collagen content in stellate cells
from non-NASH subjects (donors 1, 2). Right 3 bars refer to
stellate cells from NASH patients (donors 3-7) with fibrosis
treated with niacin at 0, 0.25, and 0.5 mM. *, p<0.001 vs 0 mM
Niacin; a, p<0.0001 vs Normal (Non-NASH subject); b, p<0.03
vs 0.25 mM Niacin.
[0007] FIG. 2 shows that niacin prevented TGF-.beta. or
H.sub.2O.sub.2-induced collagen production in human hepatic
stellate cells from human subjects without liver fibrosis. Cells
were incubated with TGF-.beta. (20 ng/mL) or H.sub.2O.sub.2 (25
.mu.M) in the absence or presence of niacin (0.5 mM) for 24 h.
Cells were stained with Sirius Red for assessment of collagen
content. Top Panel: Representative cellular photographic images of
collagen deposition. Bottom Panel: Quantitation of collagen content
in stellate cells. VEH, PBS vehicle, NIA, treatment with niacin
(0.5 mM) alone without H.sub.2O.sub.2 or TGF-.beta.. *, p<0.05
vs VEH; +, p<0.05 vs respective H.sub.2O.sub.2 or
TGF-.beta..
[0008] FIG. 3 demonstrates that niacin reversed hepatic stellate
cell fibrosis induced by TGF-.beta. or H.sub.2O.sub.2 in human
hepatic stellate cells from normal human donor subjects without
liver fibrosis. Human hepatic stellate cells from control subjects
without liver fibrosis were first stimulated with TGF-.beta. (20
ng/mL) or H.sub.2O.sub.2 (25 .mu.M) for 24 h to induce collagen
deposition. These cells were then continued to incubate additional
24 h in the absence or presence of niacin (0.5 mM). The cellular
content of Collagen type I was assessed. Control, no prior
treatment with H.sub.2O.sub.2 or TGF-.beta.; Niacin 0.5 mM,
treatment with only niacin without prior treatment with
H.sub.2O.sub.2 or TGF-.beta. *, p<0.05 vs Control; +, p<0.05
vs respective H.sub.2O.sub.2 or TGF-.beta..
[0009] FIG. 4 demonstrates that niacin prevented human hepatic
stellate cell oxidative stress induced by palmitic acid or
H.sub.2O.sub.2. Cells from subjects without liver fibrosis were
first stimulated with either palmitic acid (0.5 mM) or
H.sub.2O.sub.2 (25 .mu.M) for 24 h to induce oxidative stress.
These cells were incubated an additional 24 h in the absence or
presence of niacin (0.5 mM). The ROS levels were then measured. Top
Panel: Representative cellular photographic images after staining
with DCFDA. Bottom Panel: Quantitative levels of ROS presented as
percent control, VEH, PBS vehicle alone, NIA, treatment with niacin
(0.5 mM) alone without H.sub.2O.sub.2 or palmitic acid. PA or PAL,
palmitic acid. *, p<0.05 vs VEH; +, p<0.05 vs Palmitic acid
or H.sub.2O.sub.2, respectively.
[0010] FIG. 5 provides that niacin had no effect on human hepatic
stellate cell viability. Cells were incubated with niacin (0-0.5
mM) for 0 h, 48 h, or 96 h. Cellular viability was assessed. NIA,
niacin.
[0011] FIG. 6 diagrams a possible mechanism of action of niacin on
liver fibrosis resolution. Based on the results presented herein,
it postulated that reduction of oxidative stress is a major route
for niacin's effect on fibrosis and hence improvement in cirrhosis.
By inhibiting NADPH oxidase and glutathione peroxidase, hepatic
oxidative stress is reduced resulting in fibrosis mitigation by 2
major pathways: (1) reduced lipotoxicity deactivates stellate cells
thereby leading to reductions in TGF-.beta. and its amplifying
CTGF-mediated signaling events, and a reduction in collagen
production; and (2) a reduction in oxidative stress leading to a
decrease in TIMP activity and an increase in MMP activity. Thus, it
is proposed that niacin also reduces the TIMP/MMP ratio resulting
in accelerated degradation of collagen. The combined effect these 2
pathways is fibrosis resolution. *Demonstrated in animal NASH
fibrosis model and in human stellate cell model. .alpha.-SMA:
.alpha.-smooth muscle cell actin, TGF-.beta.: transforming growth
factor-4, CTGF: connective tissue growth factor, TIMP: tissue
inhibitor of metalloproteinase, MMP: matrix metalloproteinase
DETAILED DESCRIPTION
[0012] As used herein and in the appended claims, the singular
forms "a", "an", and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"an extracellular matrix protein" includes a plurality of such
extracellular matrix proteins and reference to "the liver disease"
includes reference to one or more liver diseases thereof known to
those skilled in the art, and so forth.
[0013] Also, the use of "or" means "and/or" unless stated
otherwise. Similarly, "comprise," "comprises," "comprising"
"include," "includes," and "including" are interchangeable and not
intended to be limiting.
[0014] It is to be further understood that where descriptions of
various embodiments use the term "comprising," those skilled in the
art would understand that in some specific instances, an embodiment
can be alternatively described using language "consisting
essentially of" or "consisting of."
[0015] All publications mentioned herein are incorporated herein by
reference in their entirety for the purposes of describing and
disclosing methodologies that might be used in connection with the
description herein. Moreover, with respect to any term that is
presented in the publications that is similar to, or identical
with, a term that has been expressly defined in this disclosure,
the definition of the term as expressly provided in this disclosure
will control in all respects.
[0016] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Although many methods and reagents similar to or equivalent to
those described herein can be used in the practice of the disclosed
methods and compositions, the exemplary methods and materials are
now described.
[0017] The term "about", as used herein, is intended to qualify the
numerical values which it modifies, denoting such a value as
variable within a margin of error. When no particular margin of
error, such as a standard deviation to a mean value given in a
chart or table of data, is recited, the term "about" should be
understood to mean that range which would encompass the recited
value and the range which would be included by rounding up or down
to that figure as well, taking into account significant
figures.
[0018] The term "disorder" as used herein is intended to be
generally synonymous, and is used interchangeably with, the terms
"disease" and "condition" (as in medical condition), in that all
reflect an abnormal condition of the human or animal body or of one
of its parts that impairs normal functioning, is typically
manifested by distinguishing signs and symptoms.
[0019] The term "high-dose niacin" or "a high dose of niacin"
refers herein to a dose of niacin or a niacin analog thereof that
is from 250 mg to 2000 mg of niacin, or niacin equivalent dosing of
a niacin analog thereof "High-dose niacin" can include a
pharmaceutical dose of niacin, or a dietary dose of niacin.
[0020] The term "pharmaceutical dose of niacin" as used herein
refers to a dose of niacin or a niacin analog thereof that is
effective to reverse or regress fibrosis and/or liver cirrhosis in
a subject. A "pharmaceutical dose of niacin" greatly exceeds the
daily recommended dietary allowance (20 mg) of niacin. For purposes
of this disclosure, a "pharmaceutical dose of niacin" refers to a
dose of niacin or niacin analog thereof that is under the care and
monitoring of a healthcare provider. Accordingly, a "pharmaceutical
dose of niacin" constitutes a pharmaceutical use, not a dietary
supplement use.
[0021] The term "niacin analog" refers to a compound that has a
structure that may differ from niacin but when administered to a
subject elucidates a similar response as niacin. A "niacin analog"
includes prodrugs of niacin, niacin metabolites, mimetics of
niacin, and derivatives of niacin. Specific examples of "niacin
analogs" include, but are not limited to, acifran, acipimox,
niceritrol, isonicotinic acid, isonicotinic hydrazide, 3-pyridine
acetic acid, 5-methylnicotinic acid, pyridazine-4-carboxylic acid,
pyrazine-2-carboxylic acid, ARI-3037MO, 3-pyridylcarbinol,
3-acetylpryidine, and nicotinamide riboside chloride. Further
examples of "niacin derivatives", "niacin analogues", and "niacin
mimetics" can be found in the following patent application, which
are incorporated in full herein: US20160151343A1, U.S. Pat. No.
9,511,060B2, U.S. Pat. No. 9,212,142B2, U.S. Pat. No. 8,937,063B2,
WO2005102331A1, RU2588133C2, AU2015203711A1, WO2012175049A1,
CN103096895B CA2659747C, AU2005272043B2, JP2008518957A, and
JP2008520715A. In a particular embodiment, the term "niacin analog"
does not include inositol hexanicotinate (IHN).
[0022] The term "niacin metabolite" or "metabolite of niacin" as
used herein refers to a metabolite generated from metabolism of
niacin by an organism, particularly a mammalian organism. Specific
examples of "niacin metabolites" include, but are not limited to,
nicotinuric acid, nicotinamide, 6-hydroxy nicotinamide,
N-methyl-nicotinamide, nicotinamide-N-oxide,
N-methyl-2-pyridone-5-carboxamide, and
N-methyl-4-pyridone-5-carboxamide.
[0023] The terms "treat", "treating", and "treatment" are meant to
include alleviating or abrogating a disorder or one or more of the
symptoms associated with a disorder; or alleviating or eradicating
the cause(s) of the disorder itself. As used herein, reference to
"treatment" of a disorder is intended to include prevention.
[0024] The terms "prevent", "preventing", and "prevention" refer to
a method of delaying or precluding the onset of a disorder; and/or
its attendant symptoms, barring a subject from acquiring a disorder
or reducing a subject's risk of acquiring a disorder.
[0025] The terms "reversing fibrosis", "regressing fibrosis", or
"resolving fibrosis" are used interchangeably herein, and refer to
a process in which the accumulation of extracellular matrix
proteins (e.g., collagen) in cells is reduced or suppressed, and/or
where the levels of extracellular matrix proteins in fibrotic
tissues or organs are decreased or degraded.
[0026] The terms "reversing liver fibrosis", "regressing liver
fibrosis", or "resolving liver fibrosis" are used interchangeably
herein, and refer to a process in which the accumulation of
extracellular matrix proteins (e.g., collagen) in stellate cells is
reduced or suppressed, and/or where the levels of extracellular
matrix proteins in fibrotic liver tissue or the liver itself is
decreased or degraded.
[0027] The term "therapeutically effective amount" refers to the
amount of a compound (e.g., high-dose niacin, or a niacin analog
thereof) that, when administered, is sufficient to prevent
development of, or alleviate to some extent, one or more of the
symptoms of the disorder being treated (e.g., fibrosis). The term
"therapeutically effective amount" also refers to the amount of a
compound that is sufficient to elicit the biological or medical
response of a cell, tissue, system, animal, or human that is being
sought by a researcher, veterinarian, medical doctor, or
clinician.
[0028] The term "subject" refers to an animal, including, but not
limited to, a primate (e.g., human, monkey, chimpanzee, gorilla,
and the like), rodents (e.g., rats, mice, gerbils, hamsters,
ferrets, and the like), lagomorphs, swine (e.g., pig, miniature
pig), equine, canine, feline, and the like. The terms "subject" and
"patient" are used interchangeably herein in reference, for
example, to a mammalian subject, such as a human patient.
[0029] The term "combination therapy" means the administration of
two or more therapeutic agents (e.g., high-dose niacin, or a niacin
analog thereof, and anti-thrombotic) to treat a therapeutic
disorder described in the present disclosure. Such administration
encompasses co-administration of these therapeutic agents in a
substantially simultaneous manner, such as in a single capsule or
tablet having a fixed ratio of active ingredients or in multiple,
separate capsules for each active ingredient. In addition, such
administration also encompasses use of each type of therapeutic
agent in a sequential manner. In either case, the treatment regimen
will provide beneficial effects of the drug combination in treating
the disorders described herein.
[0030] The term "therapeutically acceptable" refers to those
compounds (or salts, prodrugs, tautomers, zwitterionic forms, etc.)
which are suitable for use in contact with the tissues of patients
without excessive toxicity, irritation, allergic response,
immunogenicity, are commensurate with a reasonable benefit/risk
ratio, and are effective for their intended use.
[0031] The term "pharmaceutically acceptable carrier",
"pharmaceutically acceptable excipient", "physiologically
acceptable carrier", or "physiologically acceptable excipient"
refers to a pharmaceutically-acceptable material, composition, or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent, or encapsulating material. Each component must be
"pharmaceutically acceptable" in the sense of being compatible with
the other ingredients of a pharmaceutical formulation. It must also
be suitable for use in contact with the tissue or organ of humans
and animals without excessive toxicity, irritation, allergic
response, immunogenicity, or other problems or complications,
commensurate with a reasonable benefit/risk ratio. See, Remington:
The Science and Practice of Pharmacy, 21st Edition; Lippincott
Williams & Wilkins: Philadelphia, Pa., 2005; Handbook of
Pharmaceutical Excipients, 5th Edition; Rowe et al., Eds., The
Pharmaceutical Press and the American Pharmaceutical Association:
2005; and Handbook of Pharmaceutical Additives, 3rd Edition; Ash
and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical
Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca
Raton, Fla., 2004.
[0032] The terms "active ingredient", "active compound", and
"active substance" refer to a compound, which is administered,
alone or in combination with one or more pharmaceutically
acceptable excipients or carriers, to a subject for treating,
preventing, or ameliorating one or more symptoms of a disorder. In
a particular embodiment, high-dose niacin, or a niacin analog
thereof, is an active ingredient in a composition, such as a
pharmaceutical composition.
[0033] The terms "drug", or "therapeutic agent", refer to a
compound, or a pharmaceutical composition thereof, which is
administered to a subject for treating, preventing, or ameliorating
one or more symptoms of a disorder.
[0034] The term "release controlling excipient" refers to an
excipient whose primary function is to modify the duration or place
of release of the active substance from a dosage form as compared
with a conventional immediate release dosage form.
[0035] The term "non-release controlling excipient" refers to an
excipient whose primary function do not include modifying the
duration or place of release of the active substance from a dosage
form as compared with a conventional immediate release dosage
form.
[0036] Hepatic fibrosis is a scarring process associated with an
increased and altered deposition of extracellular matrix components
in the liver. It is caused by a variety of stimuli and if fibrosis
continues unopposed, it would progress to cirrhosis which poses a
significant health problem worldwide. Liver fibrosis is initiated
by a cascade of events resulting in hepatocyte damage, recruitment
of inflammatory cells to the injured liver, and activation of
collagen-producing cells. Hepatic stellate cells (HSC) are a major
source of collagen type 1. The fibrogenic response is a complex
process in which accumulation of extracellular matrix proteins,
tissue contraction, and alteration in blood flow are prominent.
Progressive scarring in response to a persisting liver insult
eventually results in cirrhosis which is one of the leading causes
of death worldwide and a major global health burden. At the
cellular and molecular level, this progressive process is
characterized by cellular activation of hepatic stellate cells and
aberrant activity of transforming growth factor-0 with its
downstream cellular mediators. Stellate cells are quiescent in
normal liver, but upon activation by liver injury these activated
hepatic stellate cells characterized by .alpha.-smooth cell actin
are the primary cell types producing extracellular matrix proteins
leading to hepatic fibrosis. Increased hepatic oxidative stress and
generation of reactive oxygen species (ROS, an index of oxidative
stress) play a crucial role in stellate cell activation and hepatic
fibrosis. Hepatic fibrosis with persistent deposition of collagen
results in distortion of hepatic parenchyma and vascular structure,
clinically manifesting as liver cirrhosis and with fatal
complications.
[0037] Liver fibrosis results from perpetuation of the normal wound
healing response, resulting in an abnormal continuation of
fibrogenesis. It is characterized by an excessive deposition of
extracellular matrix (ECM) proteins which includes three large
families of proteins--glycoproteins, collagens, and proteoglycans.
Fibrosis occurs as a result of repeated cycles of hepatocytes
injury and repair. The cascade of events that establish hepatic
fibrosis is complex, and is influenced by how different cell types
in the liver interact in response to injury, and activation of HSC
is the central event. Liver fibrosis is a dynamic process; it is
usually secondary to hepatic injury and inflammation, and
progresses at different rates depending on the etiology of liver
disease and is also influenced by environmental and genetic
factors. If fibrosis continues unopposed, it would disrupt the
normal architecture of the liver which alters the normal function
of the organ, ultimately leading to pathophysiological damage of
the liver. Cirrhosis represents the final stages of fibrosis. It is
characterized by fibrous septa which divide the parenchyma into
regenerative nodules which leads to vascular modifications and
portal hypertension with its complications of variceal bleeding,
hepatic encephalopathy, ascites, and hepatorenal syndrome. In
addition, this condition is largely associated with hepatocellular
carcinoma with a further increase in the relative mortality
rate.
[0038] Liver cirrhosis is a silent disease and clinical
manifestations of portal hypertension from cirrhosis occur after
decades of life with serious and costly complications including
ascites, esophageal varices and hemorrhage, hepatic encephalopathy,
and liver failure. Prolonged cirrhosis has been recognized as a
"premalignant state" with increased risk of hepatocellular
carcinoma. Thus, prevention in high-risk individuals is as
important as treatment. Current management emphasizes lifestyle
change (diet and exercise) as the initial treatment. However, this
is not always successful and pharmacologic approaches are
needed.
[0039] The mechanisms able to elicit and sustain liver fibrogenesis
may be classified in three main groups: (a) chronic activation of
the wound healing reaction, (b) oxidative stress, and (c)
derangement of epithelial-mesenchymal interactions and
epithelial-mesenchymal transition in cholangiopathies.
[0040] Similar to what was observed in other fibrogenic disorders
affecting different organs and systems, the chronic activation of
the wound-healing reaction is the most common and relevant
mechanism in hepatic fibrogenesis. Overall, hepatic fibrogenesis
due to the chronic activation of the wound healing reaction is
characterized by the following key features: (i) the persistence of
hepatocellular/cholangiocellular damage with variable degree of
necrosis and apoptosis; (ii) a complex inflammatory infiltrate
including mononuclear cells and cells of the immune system; (iii)
the activation of different types of ECM-producing cells (HSCs,
portal myofibroblasts (MFs), etc.) with marked proliferative,
synthetic, and contractile features; and (iv) marked changes in the
quality and quantity of the hepatic ECM associated with very
limited or absent possibilities of remodeling in the presence of a
persistent attempt of hepatic regeneration.
[0041] Involvement of oxidative stress has been documented in all
human major clinical conditions of chronic liver disease (CLD) as
well as in most experimental models of liver fibrogenesis, but it
is likely to represent the predominant profibrogenic mechanism
mainly in NAFLD/NASH and alcoholic steatohepatitis (ASH). Oxidative
stress in CLD, resulting from increased generation of reactive
oxygen species (ROS) and other reactive intermediates as well as by
decreased efficiency of antioxidant defenses, does not represent
simply as a potentially toxic consequence of chronic liver injury
but actively contributes to excessive tissue remodeling and
fibrogenesis. ROS and other reactive mediators such as
4-hydroxynonenal (HNE) can be generated outside MFs, being released
either by activated inflammatory cells or deriving from
hepatocytes, directly or indirectly, damaged by the specific
etiological agent or conditions. Indeed, oxidative stress,
presumably by favoring mitochondrial permeability transition, is
able to promote hepatocyte death (necrotic and/or apoptotic). In
some of clinically relevant conditions, generation of ROS within
hepatocytes may represent a consequence of an altered metabolic
state (like in NAFLD and NASH) or of ethanol metabolism (as in
ASH), with ROS being generated mainly by mitochondrial electron
transport chain or through the involvement of selected cytochrome
P.sub.450 isoforms like cytochrome P2E1 (CYP2E1).
Oxidative-stress-related mediators released by damaged or activated
neighboring cells can directly affect the behavior of human
HSC/MFs: ROS or the reactive aldehyde HNE have been reported to
upregulate expression of critical genes related to fibrogenesis
including procollagen type I, monocyte chemoattractant protein 1
(MCP-1), and TIMP-1, possibly through activation of a number of
critical signal transduction pathways and transcription factors,
including activation of c-jun N-terminal kinases (JNKs),
transcription factor AP-1 (AP-1) and for ROS, nuclear factor-kB
(NF-kB). In addition to `profibrogenic` extracellular release by
neighboring cells, ROS generation within human and rat HSC/MFs has
been reported to occur in response to several known profibrogenic
mediators, including angiotensin II, platelets derived growth
factor (PDGF), and the adipokine leptin.
[0042] Although several therapeutic targets and drugs have been
under investigation, there currently are no FDA approved
pharmacological agent(s) for treating liver fibrosis to reduce
portal hypertension and cirrhosis complications. It was found
herein, that high-dose niacin can be used for the treatment of
liver fibrosis and complications thereof. In the studies presented
herein high-dose niacin significantly decreased the production of
reactive oxygen species (ROS, an index of oxidative stress) via
inhibition of NADPH oxidase activity in human hepatocytes and
inflammation in various cell types including human aortic
endothelium and human blood lymphocytes. It is postulated herein
that reduction of oxidative stress and inflammation may be account,
at least partially, for fibrosis reduction by niacin.
[0043] The studies presented herein demonstrate that niacin not
only prevented and reversed collagen deposition in hepatic stellate
cells isolated from non-fibrotic livers, but also reversed
preexisting collagen deposits (fibrosis) in hepatic stellate cells
taken from patients with NASH-fibrosis. The mechanism is by
oxidative stress reduction by niacin.
[0044] Niacin, also known as nicotinic acid or vitamin B3, is a
water-soluble vitamin whose derivatives such as NADH, NAD,
NAD.sup.+, and NADP play essential roles in energy metabolism in
the living cell and DNA repair. The designation vitamin B3 also
includes the amide form, nicotinamide or niacinamide. Severe lack
of niacin causes the deficiency disease pellagra, whereas a mild
deficiency slows down the metabolism decreasing cold tolerance. The
recommended daily allowance of niacin is 2-12 mg a day for
children, 14 mg a day for women, 16 mg a day for men, and 18 mg a
day for pregnant or breast-feeding women. It is found in various
animal and plant tissues and has pellagra-curative, vasodilating,
and antilipemic properties. The liver can synthesize niacin from
the essential amino acid tryptophan, but the synthesis is extremely
slow and requires vitamin B6; 60 mg of tryptophan are required to
make one milligram of niacin. Bacteria in the gut may also perform
the conversion but are inefficient.
[0045] Both niacin and niacinamide are rapidly absorbed from the
stomach and small intestine. Absorption is facilitated by
sodium-dependent diffusion, and at higher intakes, via passive
diffusion. Unlike some other vitamins, the percent absorbed does
not decrease with increasing dose, so that even at amounts of 3-4
grams, absorption is nearly complete. With a one gram dose, peak
plasma concentrations of 15 to 30 .mu.g/mL are reached within 30 to
60 minutes. Approximately 88% of an oral pharmacologic dose is
eliminated by the kidneys as unchanged niacin or nicotinuric acid,
its primary metabolite. The plasma elimination half-life of niacin
ranges from 20 to 45 minutes.
[0046] After absorption, niacin circulates in the plasma in the
unbound form as both the acid and the amide. In the liver,
niacinamide is converted to storage nicotinamide adenine
dinucleotide (NAD). As needed, liver NAD is hydrolyzed to
niacinamide and niacin for transport to tissues. The tissues then
reconvert niacinamide and niacin to NAD to serve as an enzyme
cofactor. Excess niacin is methylated in the liver to
N.sup.1-methylnicotinamide (NMN) and excreted in urine as such or
as the oxidized metabolite N.sup.1-methyl-2-pyridone-5-carboxamide
(2-pyridone). The main metabolites in humans are
N-methylnicotinamide, N-methyl-2-pyridone-5-carboxamide,
N-methyl-6-pyridone-3-carboxamide,
N-methyl-4-pyridone-3-carboxamide and
N-methyl-4-pyridone-5-carboxamide. Decreased urinary content of
these metabolites is a measure of niacin deficiency.
[0047] Niacin is incorporated into multi-vitamin and sold as a
single-ingredient dietary supplement. The latter can be immediate
or slow release. One form of dietary supplement sold in the US is
inositol hexanicotinate (IHN), also called inositol nicotinate. IHP
is made up of inositol that has been esterified with niacin on all
six of inositol's alcohol groups. IHN is usually sold as
"flush-free" or "no-flush" niacin in units of 250, 500, or 1000
mg/tablets or capsules. In the US, it is sold as an
over-the-counter formulation, and often is marketed and labeled as
niacin, thus misleading consumers into thinking they are getting an
active form of the medication. While this form of niacin does not
cause the flushing associated with the immediate-release products,
gastrointestinal absorption of inositol hexanicotinate varies
widely with an average of 70% of an orally ingested dose absorbed.
Once inositol hexanicotinate is present in human serum, hydrolysis
of the ester bonds and release of free nicotinic acid is slow,
taking more than 48 hours. After oral doses of 0.8 to 4.2 g of
inositol hexanicotinate in humans, plasma levels of free nicotinic
acid peaks at 6-12 hours. In contrast, after an oral dose of 1000
mg of nicotinic acid, plasma levels of free nicotinic acid peaks at
0.5-1 hour at 30 .mu.g/mL. While IHN is well tolerated, it was
found to be no better than placebo for the management of
dyslipidemia (see Keenan, Joseph, "Extended-Release Nicotinic Acid
Versus Inositol Hexanicotinate for the Treatment of Dyslipidemia."
Journal of Clinical lipidology, 4(3):P216-217 (2010)). IHN does not
produce plasma nicotinic acid levels sufficient to lower lipids.
The peak plasma levels of nicotinic acid after oral doses of IHN
are dramatically lower when compared with those obtained after oral
doses of nicotinic acid; for example, a single oral dose of 1,000
mg nicotinic resulted in a peak plasma level of 30 .mu.g/mL
nicotinic acid, while 1,000 mg of IHN (weight equivalent to
.about.910 mg nicotinic acid) resulted in a peak plasma level of
0.2 .mu.g/mL nicotinic acid (see Harthon et al., "Enzymatic
hydrolysis of pentaerythritoltetranicotinate and
meso-inositolhexanicotinate in blood and tissues."
Arzneimittelforschung. 29:1859-1862 (1979)).
[0048] Quantities of niacin above 500 mg should not be
self-administered as a dietary supplement, but may be safely used
under the care and monitoring of a healthcare provider. Such an
application, it should be noted, constitutes a pharmaceutical use,
not a dietary supplement use. Niacin, when used at higher doses,
has been used clinically for the treatment of lipid disorders and
cardiovascular disease. Pharmacologic doses of niacin have been
shown to reduce atherogenic lipids, lipoproteins and several
inflammatory markers. As monotherapy, it significantly minimized
cardiovascular and stroke events and slowed or reversed occlusive
atherosclerosis in combination with LDL-C lowering agents. While
niacin has been shown to reverse hepatic steatosis and
inflammation, its efficacy on reversing or treating fibrosis in
humans is still not known.
[0049] Pharmacologic doses of niacin can be immediate release
(Niacor, 500 mg tablets) or extended release (Niaspan, 500 and 1000
mg tablets). Niaspan has a film coating that delays release of the
niacin, resulting in an absorption over a period of 8-12 hours.
Both forms of niacin are considered safe and effective
antihyperlipidemic drugs for use under medical supervision and
monitoring. Extended-release formulations of niacin substantially
reduce the risk of flushing reactions, but carry a greater risk of
liver toxicity. ER-NA is approximately twice as hepatotoxic as NA.
Combining laropiprant, a prostaglandin D2 binding drug, with niacin
leads to a reduction of niacin-induced vasodilation and flushing
side effects.
[0050] In a preclinical in vitro human model of liver fibrosis, it
was shown herein that niacin's efficacy on stellate cell fibrosis
reversal and prevention. In the study, primary stellate cells were
isolated and cultured in vitro, from fresh livers of patients who
had recently died. Patients with histologically diagnosed fibrosis
(stage 1 to stage 3) were investigated. Two patients had no
fibrosis. As shown (Table 1), these patients were of both genders,
wide age range, had comorbidities of varying etiologies including
risk factors for NASH, and racial backgrounds. Pharmacological
concentrations of niacin (0.25-0.5 mM) used in the in-vitro studies
in hepatic stellate cells are clinically relevant and comparable to
the niacin concentrations observed in human plasma after oral
administration of niacin doses of 1-3 g/daily. Because of the first
pass effect via the portal vein, niacin concentration in liver
tissue will be much higher than in plasma levels after oral
administration of 1-3 g of niacin.
[0051] The data in the study demonstrate that niacin (0.25 mM and
0.5 mM) in a dose and time dependent manner markedly (up to 65%)
decreased pre-existing fibrosis in stellate cells from patients
with varying degrees of fibrotic NASH (NAFLD activity scores 2-5,
Fibrosis scores 1-3) in a statistically significant manner. In
non-NASH subjects, niacin strikingly prevented, and regressed
stellate cell fibrosis induced by major physiological stimulators
of liver fibrosis such as inflammatory cytokine TGF-.beta. or
oxidative stress mediator H.sub.2O.sub.2.
[0052] Of note, in addition to the direct effects of niacin on
hepatic fibrosis demonstrated herein, there is also an indirect
effect via the mitigating effects of niacin on steatosis and
inflammation. By reducing the front-end of this progressive
disease, niacin reduced the sequential cascade effect on
inflammation and fibrosis. Thus, niacin is a multifactorial
therapeutic due to its direct and indirect efficacy on fibrosis
reversal and prevention.
[0053] Collagen 1 is an important component of liver fibrosis
resulting in clinical cirrhosis. The balance between its production
and removal determines its content in the liver over time.
Oxidative stress and Transforming Growth Factor-beta (TGF-.beta.)
play an important role in the pathogenesis, production, and
accumulation of hepatic collagen. Briefly, mediators of oxidative
stress including NADPH oxidase, Glutathione Peroxidase (GPx),
Hydrogen Peroxide (H.sub.2O.sub.2) and saturated fatty acids (e.g.,
palmitic acid) result in increased lipid peroxidation and
activation of hepatocytes and stellate cells as indicated by the
marker alpha-Smooth Muscle Actin (.alpha.-SMA). The production of
TGF-.beta. from oxidative stress has major impact on fibrogenesis.
Its known multiple effects include apoptosis in hepatocytes,
increased hydrogen peroxide further augmenting oxidative stress in
a vicious cycle, and increased production of Connective Tissue
Growth Factor (CTGF), a mitogenic protein that amplifies
fibrogenesis. These factors result in stimulation of extracellular
matrix proteins of which collagen 1 is most abundant. The
physiologic removal of hepatic collagen is mediated by macrophages
that secrete matrix metalloproteinases (MMP), including MMP 2 and
9. MMPs are regulated by tissue inhibitors of metalloproteinases
(TIMPs). The dynamic balance between the MMPs and TIMPs impacts on
collagen removal rate. Thus, a decrease in the TIMP/MMP ratio
either by increase of MMP or a decrease in TIMP alone or both
results in increased collagen removal.
[0054] In the studies presented herein, it was shown that high-dose
niacin significantly and robustly removed preexisting collagen in
cultured stellate cells isolated from a variety of NASH patients
with histologically demonstrated fibrosis (grade 1 to grade 3).
When human stellate cells, isolated from patients without fibrosis,
were challenged with TGF-.beta. or hydrogen peroxide, high-dose
niacin suppressed collagen deposition by over 55% (see FIG. 2).
High-dose niacin reduced oxidative stress induced by palmitic acid
or hydrogen peroxide by over half (see FIG. 4). It is postulated
herein that mechanism of action for high-dose niacin is primarily
due to a reduction of oxidative stress (see FIG. 6). A reduction of
oxidative stress leads to the suppression of lipotoxicity,
decreased hepatic and stellate cell activation, reduced TGF-.beta.
and connective tissue growth factor (CTGF) production. Reduction in
TGF-.beta. would also reduce hydrogen peroxide, NF-kB, and
apoptosis. It has been shown that oxidative stress on activated
human stellate cells leads to increases in the levels of matrix
metalloproteinase-1 (TIMP-1) and decreases in the levels of matrix
metalloproteinase-1 (MMP). In a human stellate cell line,
investigators have shown that H.sub.2O.sub.2 (major component of
reactive oxidative species (ROS)) generated by leptin, through
activation of ERK1/2 and p38 signaling, inhibited MMP mRNA and
promoter activity. Leptin also stimulated TIMP promoter activity
and TIMP mRNA expression via by MAP kinase signaling mechanisms. By
using a specific ROS inhibitor (catalase, an inhibitor of
H.sub.2O.sub.2), it was shown that the opposing expression of both
MMP and TIMP are dependent on ROS-mediated downstream signaling
processes. Accordingly, it is postulated herein that high-dose
niacin, or a niacin analog thereof, will stabilize or normalize the
expression levels of MMP and TIMP.
[0055] As shown in the results presented herein, high-dose niacin
can be used to treat, and even reverse fibrosis. Thus, high-dose
niacin, or a niacin analog thereof, can be used to treat diseases,
disorders or conditions associated with fibrosis. Fibrotic disease
can affect many organs, including liver, bone marrow, lung, kidney,
gastrointestinal tract, skin, eye, musculosketal system, and
endomyocardium, leading eventually to organ failure. Specific
examples of such diseases, disorders and conditions include, but
are not limited to, pulmonary fibrosis, such as cystic fibrosis,
idiopathic pulmonary fibrosis; radiation-induced lung injury;
post-COVID 19 fibrosis; bridging NASH; liver cirrhosis; liver
fibrosis; glial scars; arterial stiffness; arthrofibrosis; Crohn's
disease, Dupuytren's contracture; Keloids; mediastinal fibrosis;
myelofibrosis; Peyronie's disease; nephrogenic system fibrosis;
progressive massive fibrosis; retroperitoneal fibrosis;
scleroderma/systemic sclerosis; adhesive capsulitis; myocardial
fibrosis, such as interstitial fibrosis and replacement fibrosis;
inflammatory bowel disease; renal fibrosis in patients with
tubulointerstitial fibrosis; glomerulosclerosis; and chronic kidney
disease. As shown in the results herein, high-dose niacin reversed
hepatic fibrosis, in part by decreasing or degrading excess ECM
proteins in cells and tissue. The accumulation of ECM proteins in
cells and tissue is a common causation component for most fibrotic
conditions and diseases. For example, pulmonary fibrosis is
characterized by excessive deposition of collagen and other
extracellular matrices (ECM) components as well as the lungs'
inability to reconstruct the damaged alveolar epithelium, and
persistence of fibroblasts.
[0056] In a particular embodiment, the disclosure provides for the
reversal or regressing of fibrosis in a subject who has been
diagnosed with liver fibrosis and/or liver cirrhosis by using
invasive liver biopsies, and/or noninvasive screening of
biochemical markers for liver fibrosis or transient elastography.
The complete evaluation of a patient with possible liver fibrosis
requires clinical evaluation, laboratory tests, and pathological
examination. The liver biopsy is regarded as the historical `gold
standard` for diagnosis and assessment of prognosis in CLD. Two
scoring methods commonly used to stage liver fibrosis are Knodell,
and METAVIR. The Knodell and METAVIR score fibrosis from stage 0-4,
with stage 4 as cirrhosis. In a particular embodiment, the
disclosure provides for administering high-dose niacin (e.g., one
or more pharmaceutical doses of niacin) to a subject who has been
diagnosed with grade 1, grade 2, grade 3 or grade 4 liver fibrosis.
In a further embodiment, the disclosure provides for administering
high-dose niacin (e.g., one or more pharmaceutical doses of niacin)
to a subject who has been diagnosed with liver cirrhosis.
[0057] Over the past years, several noninvasive tests have become
available to assess liver fibrosis, primarily in patients with
chronic hepatitis C infection. The currently available noninvasive
tests, which are surrogate markers of liver fibrosis (direct
markers of fibrosis), such as serum hyaluronate, Type IV collagen,
matrix metalloproteinase 1 (MMP), tissue inhibitor of matrix
metalloproteinase-1 (TIMP-1), laminin, and TGF (3, have limited
accuracy for diagnosis of significant fibrosis (METAVIR>F2 or
Ishak >3). Other noninvasive tests (indirect markers of liver
fibrosis) include FibroTest-ActiTest, APRI, Forns fibrosis index,
and enhanced liver fibrosis (ELF) score. The diagnostic performance
of these indices is generally good, with a receiver operating
characteristics (ROC) curve ranging from 0.77-0.88.
[0058] FT-AT, from Biopredictive, Paris, France, is a noninvasive
blood test that combines the quantitative results of six serum
biochemical markers (alfa2-macroglobulin, haptoglobin, gamma
glutamyl transpeptidase, total bilirubin, apolipoprotein A1, and
alanine aminotransferase (ALT)) with patients' age and gender in a
patented algorithm in order to generate a measure of fibrosis and
necroinflammatory activity in the liver. FT-AT provides an accurate
measurement of bridging fibrosis and/or moderate necroinflammatory
activity with area under the receiver operating curve (AUROC)
predictive value between 0.70 and 0.80, when compared to the liver
biopsy.
[0059] Transient elastography or Fibroscan (Echosens, Paris,
France) has become available, which measures liver stiffness or
elasticity to assess liver fibrosis. The scan was developed on the
principle that livers with increasing degrees of scarring or
fibrosis have decreasing elasticity and that a shear wave
propagating through stiffer material would progress faster than in
one with more elastic material. Transient elastography is painless,
rapid, and easily performed at the bedside or in the outpatient
clinic. A recent systemic review identified twelve studies, 9 for
FibroTest (N=1,679) and 4 for Fibroscan (N=546) and the area under
the curve (AUCs) for FibroTest and Fibroscan were 0.90 (95% CI not
calculable) and 0.95 (95% CI 0.87-0.99), respectively. The combined
use of transient elastography and biochemical markers can help the
clinician decide whether a liver biopsy is necessary in some
patients, and accordingly decide who to treat with high-dose
niacin, or a niacin analog thereof.
[0060] While it may be possible for the high-dose niacin, or a
niacin analog thereof, to be administered as the raw chemical, it
is also possible to present them as a pharmaceutical composition.
Accordingly, provided herein are pharmaceutical compositions which
comprise high-dose niacin (e.g., one or more pharmaceutical doses
of niacin), or a niacin analog thereof, or one or more
pharmaceutically acceptable salts, prodrugs, or solvates thereof,
together with one or more pharmaceutically acceptable carriers
thereof and optionally one or more other therapeutic ingredients.
Proper formulation is dependent upon the route of administration
chosen. Any of the well-known techniques, carriers, and excipients
may be used as suitable and as understood in the art; e.g., in
Remington's Pharmaceutical Sciences. The pharmaceutical
compositions disclosed herein may be manufactured in any manner
known in the art, e.g., by means of conventional mixing,
dissolving, granulating, dragee-making, levigating, emulsifying,
encapsulating, entrapping or compression processes. The
pharmaceutical compositions may also be formulated as a modified
release dosage form, including delayed-, extended-, prolonged-,
sustained-, pulsatile-, controlled-, accelerated- and fast-,
targeted-, programmed-release, and gastric retention dosage forms.
These dosage forms can be prepared according to conventional
methods and techniques known to those skilled in the art (see,
Remington: The Science and Practice of Pharmacy, supra;
Modified-Release Drug Delivery Technology, Rathbone et al., Eds.,
Drugs and the Pharmaceutical Science, Marcel Dekker, Inc., New
York, N.Y., 2002; Vol. 126).
[0061] The compositions include those suitable for oral, parenteral
(including subcutaneous, intradermal, intramuscular, intravenous,
intraarticular, and intramedullary), intraperitoneal, transmucosal,
transdermal, rectal and topical (including dermal, buccal,
sublingual and intraocular) administration although the most
suitable route may depend upon for example the condition and
disorder of the recipient. The compositions may conveniently be
presented in unit dosage form and may be prepared by any of the
methods well known in the art of pharmacy. Typically, these methods
include the step of bringing into association a compound of the
subject invention or a pharmaceutically salt, prodrug, or solvate
thereof ("active ingredient") with the carrier which constitutes
one or more accessory ingredients. In general, the compositions are
prepared by uniformly and intimately bringing into association the
active ingredient with liquid carriers or finely divided solid
carriers or both and then, if necessary, shaping the product into
the desired formulation.
[0062] Formulations of high-dose niacin, or a niacin analog
thereof, disclosed herein suitable for oral administration may be
presented as discrete units such as capsules, cachets or tablets
each containing a predetermined amount of the active ingredient; as
a powder or granules; as a solution or a suspension in an aqueous
liquid or a non-aqueous liquid; or as an oil-in-water liquid
emulsion or a water-in-oil liquid emulsion. The active ingredient
may also be presented as a bolus, electuary or paste.
[0063] Pharmaceutical preparations which can be used orally include
tablets, push-fit capsules made of gelatin, as well as soft, sealed
capsules made of gelatin and a plasticizer, such as glycerol or
sorbitol. Tablets may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active ingredient
in a free-flowing form such as a powder or granules, optionally
mixed with binders, inert diluents, or lubricating, surface active
or dispersing agents. Molded tablets may be made by molding in a
suitable machine a mixture of the powdered compound moistened with
an inert liquid diluent. The tablets may optionally be coated or
scored and may be formulated so as to provide slow or controlled
release of the active ingredient therein. All formulations for oral
administration should be in dosages suitable for such
administration. The push-fit capsules can contain the active
ingredients in admixture with filler such as lactose, binders such
as starches, and/or lubricants such as talc or magnesium stearate
and, optionally, stabilizers. In soft capsules, the active
compounds may be dissolved or suspended in suitable liquids, such
as fatty oils, liquid paraffin, or liquid polyethylene glycols. In
addition, stabilizers may be added. Dragee cores are provided with
suitable coatings. For this purpose, concentrated sugar solutions
may be used, which may optionally contain gum arabic, talc,
polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium dioxide, lacquer solutions, and suitable organic solvents
or solvent mixtures. Dyestuffs or pigments may be added to the
tablets or dragee coatings for identification or to characterize
different combinations of active compound doses.
[0064] High-dose niacin (e.g., one or more pharmaceutical doses of
niacin), or a niacin analog thereof, may be formulated for
parenteral administration by injection, e.g., by bolus injection or
continuous infusion. Formulations for injection may be presented in
unit dosage form, e.g., in ampoules or in multi-dose containers,
with an added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. The formulations may be presented in
unit-dose or multi-dose containers, for example sealed ampoules and
vials, and may be stored in powder form or in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carrier, for example, saline or sterile pyrogen-free water,
immediately prior to use. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and
tablets of the kind previously described.
[0065] Formulations for parenteral administration include aqueous
and non-aqueous (oily) sterile injection solutions of the active
compounds which may contain antioxidants, buffers, bacteriostats
and solutes which render the formulation isotonic with the blood of
the intended recipient; and aqueous and non-aqueous sterile
suspensions which may include suspending agents and thickening
agents. Suitable lipophilic solvents or vehicles include fatty oils
such as sesame oil, or synthetic fatty acid esters, such as ethyl
oleate or triglycerides, or liposomes. Aqueous injection
suspensions may contain substances which increase the viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol,
or dextran. Optionally, the suspension may also contain suitable
stabilizers or agents which increase the solubility of high-dose
niacin, or a niacin analog thereof, to allow for the preparation of
highly concentrated solutions.
[0066] In addition to the formulations described previously,
high-dose niacin, or a niacin analog thereof, may also be
formulated as a depot preparation. Such long-acting formulations
may be administered by implantation (for example subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example,
high-dose niacin, or a niacin analog thereof, may be formulated
with suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0067] For buccal or sublingual administration, the compositions
may take the form of tablets, lozenges, pastilles, or gels
formulated in conventional manner. Such compositions may comprise
the active ingredient in a flavored basis such as sucrose and
acacia or tragacanth.
[0068] The pharmaceutical compositions disclosed herein may be
formulated as immediate or modified release dosage forms, including
delayed, sustained, pulsed, controlled, targeted, timed, and
programmed-release forms.
[0069] The pharmaceutical compositions may be formulated as a
suspension, solid, semi-solid, or thixotropic liquid, for
administration as an implanted depot. In one embodiment, the
pharmaceutical compositions disclosed herein are dispersed in a
solid inner matrix, which is surrounded by an outer polymeric
membrane that is insoluble in body fluids but allows the active
ingredient in the pharmaceutical compositions diffuse through.
[0070] Suitable inner matrixes include polymethylmethacrylate,
polybutylmethacrylate, plasticized or unplasticized
polyvinylchloride, plasticized nylon, plasticized
polyethyleneterephthalate, natural rubber, polyisoprene,
polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetate
copolymers, silicone rubbers, polydimethylsiloxanes, silicone
carbonate copolymers, hydrophilic polymers, such as hydrogels of
esters of acrylic and methacrylic acid, collagen, cross-linked
polyvinylalcohol, and cross-linked partially hydrolyzed polyvinyl
acetate.
[0071] Suitable outer polymeric membranes include polyethylene,
polypropylene, ethylene/propylene copolymers, ethylene/ethyl
acrylate copolymers, ethylene/vinylacetate copolymers, silicone
rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated
polyethylene, polyvinylchloride, vinylchloride copolymers with
vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer
polyethylene terephthalate, butyl rubber epichlorohydrin rubbers,
ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl
alcohol terpolymer, and ethylene/vinyloxyethanol copolymer.
[0072] The pharmaceutical compositions disclosed herein may be
formulated as a modified release dosage form. As used herein, the
term "modified release" refers to a dosage form in which the rate
or place of release of the active ingredient(s) is different from
that of an immediate dosage form when administered by the same
route. Modified release dosage forms include delayed, extended,
prolonged, sustained, timed, pulsatile, controlled, accelerated,
rapid, targeted, programmed release forms, and gastric retention
dosage forms. The pharmaceutical compositions in modified release
dosage forms can be prepared using a variety of modified release
devices and methods known to those skilled in the art, including,
but not limited to, matrix-controlled release devices, osmotic
controlled release devices, multiparticulate controlled release
devices, ion-exchange resins, enteric coatings, multilayered
coatings, microspheres, liposomes, and combinations thereof. The
release rate of the active ingredient(s) can also be modified by
varying the particle sizes and polymorphism of the active
ingredient(s).
[0073] In one embodiment, the pharmaceutical compositions disclosed
herein in a modified release dosage form is formulated using an
erodible matrix device, which is water-swellable, erodible, or
soluble polymers, including synthetic polymers, and naturally
occurring polymers and derivatives, such as polysaccharides and
proteins.
[0074] Materials useful in forming an erodible matrix include, but
are not limited to, chitin, chitosan, dextran, and pullulan; gum
agar, gum arabic, gum karaya, locust bean gum, gum tragacanth,
carrageenans, gum ghatti, guar gum, xanthan gum, and scleroglucan;
starches, such as dextrin and maltodextrin; hydrophilic colloids,
such as pectin; phosphatides, such as lecithin; alginates;
propylene glycol alginate; gelatin; collagen; and cellulosics, such
as ethyl cellulose (EC), methylethyl cellulose (MEC), carboxymethyl
cellulose (CMC), CMEC, hydroxyethyl cellulose (HEC), hydroxypropyl
cellulose (HPC), cellulose acetate (CA), cellulose propionate (CP),
cellulose butyrate (CB), cellulose acetate butyrate (CAB), CAP,
CAT, hydroxypropyl methyl cellulose (HPMC), HPMCP, HPMCAS,
hydroxypropyl methyl cellulose acetate trimellitate (HPMCAT), and
ethylhydroxy ethylcellulose (EHEC); polyvinyl pyrrolidone;
polyvinyl alcohol; polyvinyl acetate; glycerol fatty acid esters;
polyacrylamide; polyacrylic acid; copolymers of ethacrylic acid or
methacrylic acid (EUDRAGIT, Rohm America, Inc., Piscataway, N.J.);
poly(2-hydroxyethyl-methacrylate); polylactides; copolymers of
L-glutamic acid and ethyl-L-glutamate; degradable lactic
acid-glycolic acid copolymers; poly-D-(-)-3-hydroxybutyric acid;
and other acrylic acid derivatives, such as homopolymers and
copolymers of butylmethacrylate, methylmethacrylate,
ethylmethacrylate, ethylacrylate,
(2-dimethylaminoethyl)methacrylate, and
(trimethylaminoethyl)methacrylate chloride; and nanoparticles, such
as chitosan, and poly-glutamic acid.
[0075] In a matrix-controlled release system, the desired release
kinetics can be controlled, for example, via the polymer type
employed, the polymer viscosity, and the particle sizes of the
polymer and/or the active ingredient, the ratio of the active
ingredient versus the polymer, and other excipients or carriers in
the compositions.
[0076] The pharmaceutical compositions disclosed herein in a
modified release dosage form may be prepared by methods known to
those skilled in the art, including direct compression, dry or wet
granulation followed by compression, melt-granulation followed by
compression.
[0077] Preferred unit dosage formulations are those containing an
effective dose, as herein below recited, or an appropriate fraction
thereof, of the active ingredient.
[0078] High-dose niacin, or a niacin analog thereof, may be
administered at a dose of 200 mg, 250 mg, 300 mg, 350 mg, 400 mg,
450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850
mg, 900 mg, 950 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg,
1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg or a range
that includes or is between any two of the foregoing doses. The
total daily dose range for adult humans is generally from 500 mg to
6000 mg. Tablets or other forms of presentation provided in
discrete units may conveniently contain an amount of high-dose
niacin, or a niacin analog thereof, which is effective at such
dosage or as a multiple of the same, for instance, units containing
500 mg to 1000 mg, usually around 500 mg.
[0079] The amount of high-dose niacin, or a metabolite or
derivative thereof that may be combined with the carrier materials
to produce a single dosage form will vary depending upon the
subject to be treated and the particular mode of
administration.
[0080] High-dose niacin, or a niacin analog thereof, can be
administered in various modes, e.g., orally, intravenously,
transdermally or by injection. The precise amount of high-dose
niacin, or a niacin analog thereof, administered to a patient will
be the responsibility of the attendant physician. The specific dose
level for any particular patient will depend upon a variety of
factors including the activity of the specific compound employed,
the age, body weight, general health, sex, diets, time of
administration, route of administration, rate of excretion, drug
combination, the precise disorder being treated, and the severity
of the disorder being treated. Also, the route of administration
may vary depending on the disorder and its severity.
[0081] In the case wherein the patient's condition does not
improve, upon the doctor's discretion the administration of
high-dose niacin, or a niacin analog thereof, may be administered
chronically, that is, for an extended period of time, including
throughout the duration of the patient's life in order to
ameliorate or otherwise control or limit the symptoms of the
patient's disorder.
[0082] In the case wherein the patient's status does improve, upon
the doctor's discretion the administration of high-dose niacin, or
a niacin analog thereof, may be given continuously or temporarily
suspended for a certain length of time (i.e., a "drug
holiday").
[0083] Once improvement of the patient's conditions has occurred, a
maintenance dose is administered if necessary. Subsequently, the
dosage or the frequency of administration, or both, can be reduced,
as a function of the symptoms, to a level at which the improved
disorder is retained. Patients can, however, require intermittent
treatment on a long-term basis upon any recurrence of symptoms.
[0084] Disclosed herein are methods to treat or reverse the effects
of fibrosis and/or liver cirrhosis in a subject in need thereof,
comprising: administering to the subject one or more doses of a
pharmaceutical composition comprising 250 mg to 2000 mg of niacin.
In a further embodiment, the subject is administered niacin, or a
niacin analog thereof, at a total daily dose of 250 mg, 300 mg, 350
mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 750 mg, 800 mg, 900 mg,
1000 mg, 1100 mg, 1200 mg, 1250 mg, 1300 mg, 1400 mg, 1500 mg, 1600
mg, 1700 mg, 1750 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg,
2250 mg, 2300 mg, 2400 mg, 2500 mg, 2600 mg, 2700 mg, 2750 mg, 2800
mg, 2900 mg, 3000 mg, 3100 mg, 3200 mg, 3300 mg, 3400 mg, 3500 mg,
3600 mg, 3700 mg, 3800 mg, 3900 mg, 4000 mg, 4100 mg, 4200 mg, 4300
mg, 4400 mg, 4500 mg, 4600 mg, 4700 mg, 4800 mg, 4900 mg, 5000 mg,
5100 mg, 5200 mg, 5300 mg, 5400 mg, 5500 mg, 5600 mg, 5700 mg, 5800
mg, 5900 mg, 6000 mg, or a range that includes or is between any
two of the foregoing values (e.g., 250 mg to 6000 mg). In yet a
further embodiment, the subject is administered high-dose niacin,
or a niacin analog thereof, for a period of 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10
weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks,
17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23
weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks
30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36
weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks,
43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49
weeks, 50 weeks, 51 weeks, 52 weeks, or a range that includes or is
between any two of the foregoing periods of time. In yet a further
embodiment, the subject is administered high-dose niacin, or a
niacin analog thereof, for a period of at least 1 week, at least 2
weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at
least 10 weeks, at least 15 weeks, at least 20 weeks, at least
weeks 30 weeks, or at least 52 weeks. Fibrosis can affect various
tissues or organs, including but not limited to, liver, bone
marrow, lung, kidney, gastrointestinal tract, skin, eye,
musculosketal system, endomyocardium, and myocardium. In a
particular embodiment, high-dose niacin, or a niacin analog
thereof, disclosed herein is used to treat or reverse the effects
of fibrosis in the liver of a subject. Fibrosis is associated with
many disease, disorders, or conditions including, but not limited
to, cystic fibrosis, idiopathic pulmonary fibrosis,
radiation-induced lung injury, nonalcoholic steatohepatitis (NASH),
liver cirrhosis, glial scars, arterial stiffness, arthrofibrosis,
Crohn's disease, Dupuytren's contracture, Keloids, mediastinal
fibrosis, myelofibrosis, Peyronie's disease, nephrogenic system
fibrosis, progressive massive fibrosis, retroperitoneal fibrosis,
scleroderma/systemic sclerosis, adhesive capsulitis, interstitial
fibrosis, replacement fibrosis, and Inflammatory Bowel disease,
Renal fibrosis in patients with tubulointerstitial fibrosis,
glomerulosclerosis, and chronic kidney disease. In a certain
embodiment, high-dose niacin, or a niacin analog thereof, disclosed
herein is used to treat or reverse the effects of fibrosis in a
subject who has NASH or liver cirrhosis.
[0085] In a certain embodiment, a method to treat or reverse the
effects of fibrosis in a subject in need thereof, comprising:
administering to the subject one or more doses of a pharmaceutical
composition comprising 500 mg to 1500 mg of niacin, or a niacin
analog thereof, so as to cause: (1) regression or reversal of
fibrosis; (2) reduction in oxidative stress; (3) reduction in the
level of tissue inhibitor metalloproteinase-1 (TIMP-1) and increase
in the level of matrix metalloproteinase-1 (MMP); and/or reversal
and/or suppression collagen deposition in stellate cells.
[0086] High-dose niacin, or a niacin analog thereof, disclosed
herein may also be combined or used in combination with other
agents useful in the treatment of thrombosis. Or, by way of example
only, the therapeutic effectiveness of high-dose niacin, or a
niacin analog thereof, described herein may be enhanced by
administration of an adjuvant (i.e., by itself the adjuvant may
only have minimal therapeutic benefit, but in combination with
another therapeutic agent, the overall therapeutic benefit to the
patient is enhanced).
[0087] Such other agents, adjuvants, or drugs, may be administered,
by a route and in an amount commonly used therefor, simultaneously
or sequentially with high-dose niacin, or a niacin analog thereof,
as disclosed herein. When high-dose niacin, or a niacin analog
thereof, is used contemporaneously with one or more other drugs, a
pharmaceutical composition containing such other drugs in addition
to high-dose niacin, or a niacin analog thereof, disclosed herein
may be utilized, but is not required.
[0088] High-dose niacin, or a niacin analog thereof, disclosed
herein is sequentially or concurrently administered in combination
with other classes of therapeutics, including, but not limited to,
anti-fibrotic therapeutics; antivirals; gallstone solubilizing
agents; anti-retroviral agents; CYP3A inhibitors; CYP3A inducers;
protease inhibitors; adrenergic agonists; anti-cholinergics; mast
cell stabilizers; xanthines; leukotriene antagonists;
glucocorticoids treatments; local or general anesthetics;
non-steroidal anti-inflammatory agents (NSAIDs), such as naproxen;
antibacterial agents, such as amoxicillin; cholesteryl ester
transfer protein (CETP) inhibitors, such as anacetrapib;
anti-fungal agents, such as isoconazole; sepsis treatments, such as
drotrecogin-a; steroidals, such as hydrocortisone; local or general
anesthetics, such as ketamine; norepinephrine reuptake inhibitors
(NRIs) such as atomoxetine; dopamine reuptake inhibitors (DARIs),
such as methylphenidate; serotonin-norepinephrine reuptake
inhibitors (SNRIs), such as milnacipran; sedatives, such as
diazepham; norepinephrine-dopamine reuptake inhibitor (NDRIs), such
as bupropion; serotonin-norepinephrine-dopamine-reuptake-inhibitors
(SNDRIs), such as venlafaxine; monoamine oxidase inhibitors, such
as selegiline; hypothalamic phospholipids; endothelin converting
enzyme (ECE) inhibitors, such as phosphoramidon; opioids, such as
tramadol; thromboxane receptor antagonists, such as ifetroban;
potassium channel openers; thrombin inhibitors, such as hirudin;
hypothalamic phospholipids; growth factor inhibitors, such as
modulators of PDGF activity; platelet activating factor (PAF)
antagonists; anti-platelet agents, such as GPIIb/IIIa blockers
(e.g., abdximab, eptifibatide, and tirofiban), P2Y(AC) antagonists
(e.g., clopidogrel, ticlopidine and CS-747), and aspirin;
anticoagulants, such as warfarin; low molecular weight heparins,
such as enoxaparin; Factor VIIa Inhibitors and Factor Xa
Inhibitors; renin inhibitors; neutral endopeptidase (NEP)
inhibitors; vasopepsidase inhibitors (dual NEP-ACE inhibitors),
such as omapatrilat and gemopatrilat; HMG CoA reductase inhibitors,
such as pravastatin, lovastatin, atorvastatin, simvastatin, NK-104
(a.k.a. itavastatin, nisvastatin, or nisbastatin), and ZD-4522
(also known as rosuvastatin, or atavastatin or visastatin);
squalene synthetase inhibitors; fibrates; bile acid sequestrants,
such as questran; niacin; anti-atherosclerotic agents, such as ACAT
inhibitors; MTP Inhibitors; gallstone solubilizing agents, such as
ursodiol; calcium channel blockers, such as amlodipine besylate;
potassium channel activators; alpha-muscarinic agents;
beta-muscarinic agents, such as carvedilol and metoprolol;
antiarrhythmic agents; diuretics, such as chlorothlazide,
hydrochiorothiazide, flumethiazide, hydroflumethiazide,
bendroflumethiazide, methylchlorothiazide, trichioromethiazide,
polythiazide, benzothlazide, ethacrynic acid, tricrynafen,
chlorthalidone, furosenilde, musolimine, bumetanide, triamterene,
amiloride, and spironolactone; thrombolytic agents, such as tissue
plasminogen activator (tPA), recombinant tPA, streptokinase,
urokinase, prourokinase, and anisoylated plasminogen streptokinase
activator complex (APSAC); anti-diabetic agents, such as biguanides
(e.g. metformin), glucosidase inhibitors (e.g., acarbose),
insulins, meglitinides (e.g., repaglinide), sulfonylureas (e.g.,
glimepiride, glyburide, and glipizide), thiozolidinediones (e.g.
troglitazone, rosiglitazone and pioglitazone), and PPAR-gamma
agonists; mineralocorticoid receptor antagonists, such as
spironolactone and eplerenone; growth hormone secretagogues; aP2
inhibitors; phosphodiesterase inhibitors, such as PDE III
inhibitors (e.g., cilostazol) and PDE V inhibitors (e.g.,
sildenafil, tadalafil, vardenafil); protein tyrosine kinase
inhibitors; antiinflammatories; antiproliferatives, such as
methotrexate, FK506 (tacrolimus, Prograf), mycophenolate mofetil;
chemotherapeutic agents; immunosuppressants; anticancer agents and
cytotoxic agents (e.g., alkylating agents, such as nitrogen
mustards, alkyl sulfonates, nitrosoureas, ethylenimines, and
triazenes); antimetabolites, such as folate antagonists, purine
analogues, and pyrridine analogues; antibiotics, such as
anthracyclines, bleomycins, mitomycin, dactinomycin, and
plicamycin; enzymes, such as L-asparaginase; farnesyl-protein
transferase inhibitors; hormonal agents, such as glucocorticoids
(e.g., cortisone), estrogens/antiestrogens,
androgens/antiandrogens, progestins, and luteinizing
hormone-releasing hormone anatagonists, and octreotide acetate;
microtubule-disruptor agents, such as ecteinascidins;
microtubule-stablizing agents, such as pacitaxel, docetaxel, and
epothilones A-F; plant-derived products, such as vinca alkaloids,
epipodophyllotoxins, and taxanes; and topoisomerase inhibitors;
prenyl-protein transferase inhibitors; and cyclosporins; steroids,
such as prednisone and dexamethasone; cytotoxic drugs, such as
azathiprine and cyclophosphamide; TNF-alpha inhibitors, such as
tenidap; anti-TNF antibodies or soluble TNF receptor, such as
etanercept, rapamycin, and leflunimide; and cyclooxygenase-2
(COX-2) inhibitors, such as celecoxib and rofecoxib; and
miscellaneous agents such as, hydroxyurea, procarbazine, mitotane,
hexamethylmelamine, gold compounds, platinum coordination
complexes, such as cisplatin, satraplatin, and carboplatin;
galectin inhibitors TGF-beta inhibitors, anti-TGF-beta antibodies,
anti-oxidants, oxidative stress inhibitors, TIMP activators, TIMP
inhibitors, MMP activators, MMP inhibitors, kynurenic acid, FS2,
cenicriviroc, aramchol, aramchol meglumine, and belapectin.
[0089] In another embodiment, high-dose niacin, or a niacin analog
thereof, disclosed herein is sequentially or concurrently
administered in combination with one or more therapeutics that
target lipid metabolism and insulin resistance; one or more
therapeutics that target lipotoxicity, oxidative stress, and
inflammation; one or more therapeutics that target fibrosis and
cirrhosis; or a combination thereof. In a certain embodiment,
high-dose niacin, or a niacin analog thereof, disclosed herein can
be administered in combination of one or more therapeutics that
target lipid metabolism and insulin resistance. Examples of such
therapeutics, include but are not limited to, acetyl-CoA
carboxylase (ACC) Inhibitors, fatty acid synthase inhibitors,
icosbutate, eicosapentaenoic acid analogs/derivatives, omega/n-3
fatty acids, thiazolidinediones (TZDs),
3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase)
inhibitors, dibrates, peroxisome proliferated-activated receptors
(PPAR)-alpha/beta/gamma/delta agonists/modulators, fibroblast
growth factor (FGF)-19/21 analogs/modulators, glucagon-like peptide
receptors (GLP-1) analogs/mimetics/modulators, ketohexokinase
inhibitors, mitochondrial pyruvate carrier inhibitor/modulators,
sodium-glucose cotransporters (SGLTs) inhibitors/modulators,
adenosine monophosphate-activated protein kinase (AMPK)
activators/modulators, stearoyl-CoA dehydrogenase (SCD) inhibitors,
diacylglycerol-acyl transferase-2 (DGAT-2) inhibitors, thyroid
hormone receptor-beta (THR-beta) agonists, glucocorticoid
modulators, dipeptidyl peptidase-4 inhibitors (DPP-4), sodium
glucose co-transporter 2 (SGLT2) inhibitors and other anti-diabetes
2 agents. In a further embodiment, high-dose niacin, or a niacin
analog thereof, disclosed herein can be administered in combination
of one or more therapeutics that target lipotoxicity, oxidative
stress, and inflammation. Examples of such therapeutics, include
but are not limited to, PPAR-alpha/beta/gamma/delta
agonists/modulators, farnesoid X receptor (FXR)
agonists/modulators, vitamin E, anti-oxidants, FGF-19/21
modulators, chemokine 2/5 receptor (CCR2/5) antagonists/Inhibitors,
nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase)
inhibitors, and angiotensin receptor antagonists. In yet another
embodiment, high-dose niacin, or a niacin analog thereof, disclosed
herein can be administered in combination of one or more
therapeutics that target fibrosis and cirrhosis. Examples of such
therapeutics, include but are not limited to, caspases
inhibitors/modulators, apoptosis inhibitors/modulators,
leukotriene/phosphodiesterase/lipoxygenase
antagonists/inhibitors/modulators, galectin-3
antagonists/modulators, apoptosis signal-regulating kinases (ASK)
inhibitors, lysophosphatidic acid receptor 1 (LPA1) antagonists,
and heat shock proteins (HSP47 and other members) inhibitors.
[0090] In a certain embodiment, high-dose niacin, or a niacin
analog thereof, disclosed herein is sequentially or concurrently
administered in combination with one or more therapeutics selected
from cenicriviroc, resmetirom, ocaliva, elafibranor, aramchol,
IMM124E, semaglutide, lanifibranor, seladelpar, belapectin,
PXL_065, MADC_0602, aldafermin, VK2809, EDP_305, PF_05221304,
tipelukast, tropifexor, DF102, LMB763, nitazoxanide, tesamorelin,
TERN_101, lazarotide, BMS986036, Saroglitazar, AKR001, CRV431,
GRI_0621, EYP001, BMS_986171, isosabutate, PF_06835919,
PF_06865571, nalmefene, LIK066, BIO89_100, Namodenoson, MT_3995,
pemafibrate, PXL770, gemcabene, foralumab, SGM_1019, KBP_042,
hepastem, CER_209, DUR928, sotagliflozin, elobixibat, SAR425899,
NGM313, namacizumab, TERN_201, LPCN_1144, ND_L02_S0201, RTU_1096,
IONIS_DGAT2R, bezafibrate, INT_767, NP160, NEULIV, NP135,
BFKB8488A, NC_001, VK0214, HM15211, CM_101, AZD2693, NV556,
SP_1373, RLBN1127, RYI_018, NVP022, VPR_423, CB4209-CB4211, and
GKT_137831. Thus, in another aspect, certain embodiments provide
methods for treating fibrosis-mediated disorders (e.g., liver
cirrhosis) in a subject in need of such treatment comprising
administering to said subject an amount of high-dose niacin, or a
niacin analog thereof, disclosed herein effective to reduce or
prevent said disorder in the subject, in combination with at least
one additional agent for the treatment of said disorder. In a
related aspect, certain embodiments provide therapeutic
compositions comprising high-dose niacin, or a niacin analog
thereof, disclosed herein in combination with one or more
additional agents for the treatment of fibrosis-mediated disorders
(e.g., liver cirrhosis).
[0091] For use in the therapeutic or biological applications
described herein, kits and articles of manufacture are also
described herein. Such kits can comprise a carrier, package, or
container that is compartmentalized to receive one or more
containers such as vials, tubes, and the like, each of the
container(s) comprising one of the separate elements to be used in
a method described herein. Suitable containers include, for
example, bottles, vials, syringes, and test tubes. The containers
can be formed from a variety of materials such as glass or
plastic.
[0092] For example, the container(s) can comprise high-dose niacin
disclosed herein, optionally in a composition or in combination
with another agent (e.g., antifibrotic therapeutic or a
prostaglandin D2 binding drug) as disclosed herein. The
container(s) optionally have a sterile access port (for example the
container can be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). Such kits
optionally comprise an identifying description or label or
instructions relating to its use in the methods described
herein.
[0093] A kit will typically comprise one or more additional
containers, each with one or more of various materials (such as
reagents, optionally in concentrated form, and/or devices)
desirable from a commercial and user standpoint for use of a
compound described herein. Non-limiting examples of such materials
include, but are not limited to, buffers, diluents, filters,
needles, syringes; carrier, package, container, vial and/or tube
labels listing contents and/or instructions for use, and package
inserts with instructions for use. A set of instructions will also
typically be included.
[0094] A label can be on or associated with the container. A label
can be on a container when letters, numbers or other characters
forming the label are attached, molded or etched into the container
itself, a label can be associated with a container when it is
present within a receptacle or carrier that also holds the
container, e.g., as a package insert. A label can be used to
indicate that the contents are to be used for a specific
therapeutic application. The label can also indicate directions for
use of the contents, such as in the methods described herein. These
other therapeutic agents may be used, for example, in the amounts
indicated in the Physicians' Desk Reference (PDR) or as otherwise
determined by one of ordinary skill in the art.
[0095] The disclosure further provides that the methods and
compositions described herein can be further defined by the
following aspects (aspects 1 to 52):
[0096] 1. A method to reverse or regress fibrosis and/or liver
cirrhosis in a subject in need thereof, comprising:
[0097] administering to a subject having fibrosis and/or liver
cirrhosis one or more pharmaceutical doses of a pharmaceutical
composition comprising niacin, or of a niacin analog thereof,
wherein the pharmaceutical composition comprises 250 mg to 2000 mg
of niacin, or niacin equivalent dosing of a niacin analog
thereof,
[0098] wherein the subject is administered a total daily dose of
250 mg to 6000 mg of niacin, or niacin equivalent dosing of a
niacin analog thereof, and
[0099] wherein administration of the one or more pharmaceutical
doses of niacin or a niacin analog thereof reverses or regresses
fibrosis and/or liver cirrhosis in the subject.
[0100] 2. The method of aspect 1, wherein the fibrosis is
associated with elevated or overaccumulation of collagen in cells
or tissue.
[0101] 3. The method of aspect 1 or aspect 2, wherein
administration of one or more pharmaceutical doses of niacin or of
a niacin analog to the subject reduces collagen levels in fibrotic
tissue.
[0102] 4. The method of any one of the preceding aspects, wherein
administration of one or more pharmaceutical doses of niacin or of
a niacin analog stabilizes or normalizes the expression levels of
matrix metalloproteinases (MMPs) and/or tissue inhibitors of
metalloproteinases (TIMPs).
[0103] 5. The method of any one of the preceding aspects, wherein
the fibrosis affects one or more tissues or organs.
[0104] 6. The method of aspect 5, wherein the one or more tissues
or organs are selected from liver, bone marrow, lung, kidney,
gastrointestinal tract, skin, eye, endomyocardium, musculoskeletal
system, and myocardium.
[0105] 7. The method of aspect 6, wherein the one or more tissues
or organs is the liver.
[0106] 8. The method of any one of the preceding aspects, wherein
the subject has a disease, disorder, or condition selected from the
group consisting of a cystic fibrosis, idiopathic pulmonary
fibrosis, post COVID-19 fibrosis, radiation-induced lung injury,
liver fibrosis, liver cirrhosis, glial scars, arterial stiffness,
arthrofibrosis, Crohn's disease, Dupuytren's contracture, keloids,
mediastinal fibrosis, myelofibrosis, Peyronie's disease,
nephrogenic system fibrosis, progressive massive fibrosis,
retroperitoneal fibrosis, scleroderma/systemic sclerosis, adhesive
capsulitis, interstitial fibrosis, replacement fibrosis,
inflammatory bowel disease, renal fibrosis in patients with
tubulointerstitial fibrosis, glomerulosclerosis, lung fibrosis, and
chronic kidney disease.
[0107] 9. The method of any one of the preceding aspects, wherein
the subject has grade 1, grade 2, grade 3, or grade 4 liver
fibrosis.
[0108] 10. The method of aspect 9, wherein the subject has grade 1,
grade 2, or grade 3 liver fibrosis.
[0109] 11. The method of aspect 9, wherein the subject has grade 4
liver fibroses (i.e., cirrhosis).
[0110] 12. The method of any one of the preceding aspects, wherein
the subject has liver fibrosis and nonalcoholic steatohepatitis, or
liver fibrosis and alcoholic steatohepatitis.
[0111] 13. The method of any one of aspects 1 to 11, wherein the
subject has liver fibrosis resulting from a biliary obstruction,
iron overload, autoimmune hepatitis, Wilson's disease, a viral
hepatitis B infection, or a viral hepatitis C infection.
[0112] 14. The method of any one of the preceding aspects, wherein
the niacin analog is selected from nicotinamide, 6-hydroxy
nicotinamide, N-methyl-nicotinamide, acifran, acipimox, niceritrol,
ARI-3037MO, and nicotinamide riboside chloride.
[0113] 15. The method of any one of the preceding aspects, wherein
the pharmaceutical composition is formulated for oral, transdermal
or parenteral delivery.
[0114] 16. The method of aspect 15, wherein the pharmaceutical
composition is formulated as an extended-release or time-release
formulation for oral delivery.
[0115] 17. The method of aspect 16, wherein the pharmaceutical
composition is formulated as a film-coated extended-release
tablet.
[0116] 18. The method of aspect 17, wherein the film-coated
extended-release tablet comprises hypromellose, povidone, stearic
acid, polyethylene glycol, and/or coloring reagents.
[0117] 19. The method of aspect 15, wherein the pharmaceutical
composition is formulated as a tablet and comprises croscarmellose
sodium, hydrogenated vegetable oil, magnesium stearate and/or
microcrystalline cellulose.
[0118] 20. The method of any one of the preceding aspects, wherein
the one or more pharmaceutical doses are administered sequentially
or concurrently with one or more therapeutics selected from
anti-fibrotic therapeutics, prostaglandin D2 binding drugs,
antivirals, gallstone solubilizing agents, anti-thrombotic
treatments, nonalcoholic fatty liver disease (NAFLD) treatments,
nonalcoholic steatohepatitis (NASH) treatments, sepsis treatments,
anti-mycobacterial agents, chelation therapy agents, anti-bacterial
agents, anti-fungal agents, steroidal drugs, anticoagulants,
non-steroidal anti-inflammatory agents, antiplatelet agents,
norepinephrine reuptake inhibitors (NRIs), dopamine reuptake
inhibitors (DRIs), Serotonin and norepinephrine reuptake inhibitors
(SNRIs), sedatives, Norepinephrine and Dopamine Reuptake Inhibitors
(NDRIs), serotonin-norepinephrine-dopamine reuptake inhibitors
(SNDRIs), monoamine oxidase inhibitors, hypothalamic phospholipids,
Endothelin converting enzymes (ECE) inhibitors, opioids,
thromboxane receptor antagonists, potassium channel openers,
thrombin inhibitors, hypothalamic phospholipids, growth factor
inhibitors, anti-platelet agents, P2Y(AC) antagonists,
anticoagulants, low molecular weight heparins, Factor VIa
Inhibitors and Factor Xa Inhibitors, renin inhibitors, neutral
endopeptidase (NEP) inhibitors, vasopepsidase inhibitors, HMG CoA
reductase inhibitors, squalene synthetase inhibitors, fibrates,
bile acid sequestrants, anti-atherosclerotic agents, microsomal
triglyceride transfer protein (MTP) Inhibitors, calcium channel
blockers, potassium channel activators, alpha-muscarinic agents,
beta-muscarinic agents, antiarrhythmic agents, diuretics,
thrombolytic agents, anti-diabetic agents, mineralocorticoid
receptor antagonists, growth hormone secretagogues, aP2 inhibitors,
phosphodiesterase inhibitors, protein tyrosine kinase inhibitors,
anti-inflammatories, anti-proliferatives, chemotherapeutic agents,
immunosuppressants, anticancer agents and cytotoxic agents,
anti-metabolites, antibiotics, farnesyl-protein transferase
inhibitors, hormonal agents, microtubule-disruptor agents,
microtubule-stabilizing agents, plant-derived products,
epipodophyllotoxins, taxanes, topoisomerase inhibitors,
prenyl-protein transferase inhibitors, cyclosporins, cytotoxic
drugs, tumor necrosis factor (TNF)-alpha inhibitors, anti-TNF
antibodies and soluble TNF receptors, cyclooxygenase-2 (COX-2)
inhibitors, galectin inhibitors, transforming growth factor
(TGF)-beta inhibitors, anti-TGF-beta antibodies, anti-oxidants,
oxidative stress inhibitors, TIMP inhibitors, matrix
metalloproteinase-1 (MMP) activators, kynurenic acid, FS2,
cenicriviroc, aramchol, aramchol meglumine, and belapectin.
[0119] 21. The method of aspect 20, wherein the one or more
pharmaceutical doses are administered sequentially or concurrently
with one or more anti-fibrotic therapeutics.
[0120] 22. The method of aspect 21, wherein the one or more
anti-fibrotic therapeutics are selected from nintedanib,
pirfenidone, rilonacept, tocilizumab, rituximab, abatacept,
lanifibranor, NCT02503644, NCT03597933, FCX-103, and SAR100842.
[0121] 23. The method of aspect 20, wherein the one or more
pharmaceutical doses is administered sequentially or concurrently
with a prostaglandin D2 binding drug.
[0122] 24. The method of aspect 23, wherein the prostaglandin D2
binding drug is laropiprant.
[0123] 25. The method of aspect 20, wherein the one or more
pharmaceutical doses are administered sequentially or concurrently
with one or more therapeutics selected from NASH treatments, NAFLD
treatments, antiviral drugs, and gallstone solubilizing agents.
[0124] 26. The method of aspect 25, wherein the NASH treatments and
NAFLD treatments are selected from orlistat, elafibranor,
pioglitazone, saroglitazar, solithromycin, exenatide, liraglutide,
sitagliptin, vildapliptin, aramchol, obeticholic acid,
cenicriviroc, pentoxifylline, emricasan, simtuzumab, galectin-3,
atorvastatin, pravastatin, cerivastatin, lovastatin, mevastatin,
pitavastatin, rosuvastatin, simvastatin, fluvastatin, NGM-282,
GS-4997, IMM-124e, cysteamine, cystamine, and vitamin E.
[0125] 27. The method of any one of the preceding aspects, wherein
the one or more pharmaceutical doses are administered sequentially
or concurrently with one or more therapeutics that target lipid
metabolism and insulin resistance; one or more therapeutics that
target lipotoxicity, oxidative stress, and inflammation; one or
more therapeutics that target fibrosis and cirrhosis; or a
combination thereof.
[0126] 28. The method of aspect 27, wherein the one or more
pharmaceutical doses are administered sequentially or concurrently
with one or more therapeutics selected from acetyl-CoA carboxylase
(ACC) Inhibitors, fatty acid synthase inhibitors, icosbutate,
eicosapentaenoic acid analogs/derivatives, omega/n-3 fatty acids,
thiazolidinediones (TZDs), 3-hydroxy-3-methylglutaryl-coenzyme A
reductase (HMG-CoA reductase) inhibitors, dibrates, peroxisome
proliferated-activated receptors (PPAR)-alpha/beta/gamma/delta
agonists/modulators, fibroblast growth factor (FGF)-19/21
analogs/modulators, glucagon-like peptide receptors (GLP-1)
analogs/mimetics/modulators, ketohexokinase inhibitors,
mitochondrial pyruvate carrier inhibitor/modulators, sodium-glucose
cotransporters (SGLTs) inhibitors/modulators, adenosine
monophosphate-activated protein kinase (AMPK)
activators/modulators, stearoyl-CoA dehydrogenase (SCD) inhibitors,
diacylglycerol-acyl transferase-2 (DGAT-2) inhibitors, thyroid
hormone receptor-beta (THR-beta) agonists, glucocorticoid
modulators, dipeptidyl peptidase-4 inhibitors (DPP-4), sodium
glucose co-transporter 2 (SGLT2) inhibitors, anti-diabetes 2
agents, PPAR-alpha/beta/gamma/delta agonists/modulators, farnesoid
X receptor (FXR) agonists/modulators, vitamin E, anti-oxidants,
FGF-19/21 modulators, chemokine 2/5 receptor (CCR2/5)
antagonists/Inhibitors, nicotinamide adenine dinucleotide phosphate
oxidase (NADPH oxidase) inhibitors, and angiotensin receptor
antagonists, caspases inhibitors/modulators, apoptosis
inhibitors/modulators, leukotriene/phosphodiesterase/lipoxygenase
antagonists/inhibitors/modulators, galectin-3
antagonists/modulators, apoptosis signal-regulating kinases (ASK)
inhibitors, lysophosphatidic acid receptor 1 (LPA1) antagonists,
and heat shock proteins (HSP47 and other members) inhibitors.
[0127] 29. The method of any one of the preceding aspects, wherein
the one or more pharmaceutical doses are administered sequentially
or concurrently with one or more therapeutics selected from
laropiprant, cenicriviroc, resmetirom, ocaliva, elafibranor,
aramchol, IMM124E, semaglutide, lanifibranor, seladelpar,
belapectin, PXL_065, MADC_0602, aldafermin, VK2809, EDP_305,
PF_05221304, tipelukast, tropifexor, DF102, LMB763, nitazoxanide,
tesamorelin, TERN_101, lazarotide, BMS986036, Saroglitazar, AKR001,
CRV431, GRI_0621, EYP001, BMS_986171, isosabutate, PF_06835919,
PF_06865571, nalmefene, LIK066, BIO89_100, Namodenoson, MT_3995,
pemafibrate, PXL770, gemcabene, foralumab, SGM_1019, KBP_042,
hepastem, CER_209, DUR928, sotagliflozin, elobixibat, SAR425899,
NGM313, namacizumab, TERN_201, LPCN_1144, ND_L02_S0201, RTU_1096,
IONIS_DGAT2R, bezafibrate, INT_767, NP160, NEULIV, NP135,
BFKB8488A, NC_001, VK0214, HM15211, CM_101, AZD2693, NV556,
SP_1373, RLBN1127, RYI_018, NVP022, VPR_423, CB4209-CB4211, and
GKT_137831.
[0128] 30. The method of any one of the preceding aspects, wherein
the subject is administered a total daily dose of 1000 mg to 3000
mg of niacin, or niacin equivalent dosing of a niacin analog
thereof.
[0129] 31. A method to suppress or inhibit accumulation of
extracellular matrix components in a subject's cells due to
inflammation, comprising:
[0130] administering to the subject one or more pharmaceutical
doses of a pharmaceutical composition comprising niacin, or of a
niacin analog thereof, wherein the composition comprises 250 mg to
2000 mg of niacin, or niacin equivalent dosing of a niacin analog
thereof,
[0131] wherein the subject is administered a total daily dose of
250 mg to 6000 mg of niacin, or niacin equivalent dosing of a
niacin analog thereof, and
[0132] wherein administration of the one or more pharmaceutical
doses of niacin or a niacin analog thereof suppresses or inhibits
accumulation of extracellular matrix components in a subject's
cells.
[0133] 32. The method of aspect 31, wherein the subject has chronic
inflammation.
[0134] 33. The method of aspect 32, wherein the chronic
inflammation is caused by exposure to environmental toxins or
pollutants, alcohol abuse, radiation treatment, medications,
medical conditions, immune diseases or disorders, cholestatic
disorders, inherited metabolic disorders, and infectious
agents.
[0135] 34. The method of any one of aspects 31 to 33, wherein the
subject's cells are hepatic stellate cells.
[0136] 35. The method of any one of aspects 31 to 34, wherein the
extracellular matrix components comprise collagen.
[0137] 36. The method of any one of aspects 31 to 35, wherein the
niacin analog is selected from nicotinamide, 6-hydroxy
nicotinamide, N-methyl-nicotinamide, acifran, acipimox, niceritrol,
ARI-3037M0, and nicotinamide riboside chloride.
[0138] 37. The method of any one of aspects 31 to 36, wherein the
pharmaceutical composition is formulated for oral, transdermal or
parenteral delivery.
[0139] 38. The method of aspect 37, wherein the pharmaceutical
composition is formulated as an extended-release or time-release
formulation for oral delivery.
[0140] 39. The method of aspect 38, wherein the pharmaceutical
composition is formulated as a film-coated extended-release
tablet.
[0141] 40. The method of aspect 39, wherein the film-coated
extended-release tablet comprises hypromellose, povidone, stearic
acid, polyethylene glycol, and/or coloring reagents.
[0142] 41. The method of aspect 37, wherein the pharmaceutical
composition is formulated as a tablet and comprises croscarmellose
sodium, hydrogenated vegetable oil, magnesium stearate and/or
microcrystalline cellulose.
[0143] 42. The method of any one of aspects 31 to 41, wherein the
one or more pharmaceutical doses are administered sequentially or
concurrently with one or more therapeutics selected from
anti-fibrotic therapeutics, prostaglandin D2 binding drugs,
antivirals, gallstone solubilizing agents, anti-thrombotic
treatments, nonalcoholic fatty liver disease (NAFLD) treatments,
nonalcoholic steatohepatitis (NASH) treatments, sepsis treatments,
anti-mycobacterial agents, chelation therapy agents, anti-bacterial
agents, anti-fungal agents, steroidal drugs, anticoagulants,
non-steroidal anti-inflammatory agents, antiplatelet agents,
norepinephrine reuptake inhibitors (NRIs), dopamine reuptake
inhibitors (DRIs), Serotonin and norepinephrine reuptake inhibitors
(SNRIs), sedatives, Norepinephrine and Dopamine Reuptake Inhibitors
(NDRIs), serotonin-norepinephrine-dopamine reuptake inhibitors
(SNDRIs), monoamine oxidase inhibitors, hypothalamic phospholipids,
Endothelin converting enzymes (ECE) inhibitors, opioids,
thromboxane receptor antagonists, potassium channel openers,
thrombin inhibitors, hypothalamic phospholipids, growth factor
inhibitors, anti-platelet agents, P2Y(AC) antagonists,
anticoagulants, low molecular weight heparins, Factor VIa
Inhibitors and Factor Xa Inhibitors, renin inhibitors, neutral
endopeptidase (NEP) inhibitors, vasopepsidase inhibitors, HMG CoA
reductase inhibitors, squalene synthetase inhibitors, fibrates,
bile acid sequestrants, anti-atherosclerotic agents, microsomal
triglyceride transfer protein (MTP) Inhibitors, calcium channel
blockers, potassium channel activators, alpha-muscarinic agents,
beta-muscarinic agents, antiarrhythmic agents, diuretics,
thrombolytic agents, anti-diabetic agents, mineralocorticoid
receptor antagonists, growth hormone secretagogues, aP2 inhibitors,
phosphodiesterase inhibitors, protein tyrosine kinase inhibitors,
anti-inflammatories, anti-proliferatives, chemotherapeutic agents,
immunosuppressants, anticancer agents and cytotoxic agents,
anti-metabolites, antibiotics, farnesyl-protein transferase
inhibitors, hormonal agents, microtubule-disruptor agents,
microtubule-stabilizing agents, plant-derived products,
epipodophyllotoxins, taxanes, topoisomerase inhibitors,
prenyl-protein transferase inhibitors, cyclosporins, cytotoxic
drugs, tumor necrosis factor (TNF)-alpha inhibitors, anti-TNF
antibodies and soluble TNF receptors, cyclooxygenase-2 (COX-2)
inhibitors, galectin inhibitors, transforming growth factor
(TGF)-beta inhibitors, anti-TGF-beta antibodies, anti-oxidants,
oxidative stress inhibitors, TIMP inhibitors, matrix
metalloproteinase-1 (MMP) activators, kynurenic acid, FS2,
cenicriviroc, aramchol, aramchol meglumine, and belapectin.
[0144] 43. The method of aspect 42, wherein the one or more
pharmaceutical doses are administered sequentially or concurrently
with one or more anti-fibrotic therapeutics.
[0145] 44. The method of aspect 43, wherein the one or more
anti-fibrotic therapeutics are selected from nintedanib,
pirfenidone, rilonacept, tocilizumab, rituximab, abatacept,
lanifibranor, NCT02503644, NCT03597933, FCX-103, and SAR100842.
[0146] 45. The method of aspect 42, wherein the one or more
pharmaceutical doses is administered sequentially or concurrently
with a prostaglandin D2 binding drug.
[0147] 46. The method of aspect 45, wherein the prostaglandin D2
binding drug is laropiprant.
[0148] 47. The method of aspect 42, wherein the one or more
pharmaceutical doses are administered sequentially or concurrently
with one or more therapeutics selected from NASH treatments, NAFLD
treatments, antiviral drugs, and gallstone solubilizing agents.
[0149] 48. The method of aspect 47, wherein the NASH treatments and
NAFLD treatments are selected from orlistat, elafibranor,
pioglitazone, saroglitazar, solithromycin, exenatide, liraglutide,
sitagliptin, vildapliptin, aramchol, obeticholic acid,
cenicriviroc, pentoxifylline, emricasan, simtuzumab, galectin-3,
atorvastatin, pravastatin, cerivastatin, lovastatin, mevastatin,
pitavastatin, rosuvastatin, simvastatin, fluvastatin, NGM-282,
GS-4997, IMM-124e, cysteamine, cystamine, and vitamin E.
[0150] 49. The method of any one of aspects 31 to 48, wherein the
one or more pharmaceutical doses are administered sequentially or
concurrently with one or more therapeutics that target lipid
metabolism and insulin resistance; one or more therapeutics that
target lipotoxicity, oxidative stress, and inflammation; one or
more therapeutics that target fibrosis and cirrhosis; or a
combination thereof.
[0151] 50. The method of aspect 49, wherein the one or more
pharmaceutical doses are administered sequentially or concurrently
with one or more therapeutics selected from acetyl-CoA carboxylase
(ACC) Inhibitors, fatty acid synthase inhibitors, icosbutate,
eicosapentaenoic acid analogs/derivatives, omega/n-3 fatty acids,
thiazolidinediones (TZDs), 3-hydroxy-3-methylglutaryl-coenzyme A
reductase (HMG-CoA reductase) inhibitors, dibrates, peroxisome
proliferated-activated receptors (PPAR)-alpha/beta/gamma/delta
agonists/modulators, fibroblast growth factor (FGF)-19/21
analogs/modulators, glucagon-like peptide receptors (GLP-1)
analogs/mimetics/modulators, ketohexokinase inhibitors,
mitochondrial pyruvate carrier inhibitor/modulators, sodium-glucose
cotransporters (SGLTs) inhibitors/modulators, adenosine
monophosphate-activated protein kinase (AMPK)
activators/modulators, stearoyl-CoA dehydrogenase (SCD) inhibitors,
diacylglycerol-acyl transferase-2 (DGAT-2) inhibitors, thyroid
hormone receptor-beta (THR-beta) agonists, glucocorticoid
modulators, dipeptidyl peptidase-4 inhibitors (DPP-4), sodium
glucose co-transporter 2 (SGLT2) inhibitors, anti-diabetes 2
agents, PPAR-alpha/beta/gamma/delta agonists/modulators, farnesoid
X receptor (FXR) agonists/modulators, vitamin E, anti-oxidants,
FGF-19/21 modulators, chemokine 2/5 receptor (CCR2/5)
antagonists/Inhibitors, nicotinamide adenine dinucleotide phosphate
oxidase (NADPH oxidase) inhibitors, and angiotensin receptor
antagonists, caspases inhibitors/modulators, apoptosis
inhibitors/modulators, leukotriene/phosphodiesterase/lipoxygenase
antagonists/inhibitors/modulators, galectin-3
antagonists/modulators, apoptosis signal-regulating kinases (ASK)
inhibitors, lysophosphatidic acid receptor 1 (LPA1) antagonists,
and heat shock proteins (HSP47 and other members) inhibitors.
[0152] 51. The method of any one of aspects 31 to 49, wherein the
one or more pharmaceutical doses are administered sequentially or
concurrently with one or more therapeutics selected from
laropiprant, cenicriviroc, resmetirom, ocaliva, elafibranor,
aramchol, IMM124E, semaglutide, lanifibranor, seladelpar,
belapectin, PXL_065, MADC_0602, aldafermin, VK2809, EDP_305,
PF_05221304, tipelukast, tropifexor, DF102, LMB763, nitazoxanide,
tesamorelin, TERN_101, lazarotide, BMS986036, Saroglitazar, AKR001,
CRV431, GRI_0621, EYP001, BMS_986171, isosabutate, PF_06835919,
PF_06865571, nalmefene, LIK066, BIO89_100, Namodenoson, MT_3995,
pemafibrate, PXL770, gemcabene, foralumab, SGM_1019, KBP_042,
hepastem, CER_209, DUR928, sotagliflozin, elobixibat, SAR425899,
NGM313, namacizumab, TERN_201, LPCN_1144, ND_L02_S0201, RTU_1096,
IONIS_DGAT2R, bezafibrate, INT_767, NP160, NEULIV, NP135,
BFKB8488A, NC_001, VK0214, HM15211, CM_101, AZD2693, NV556,
SP_1373, RLBN1127, RYI_018, NVP022, VPR_423, CB4209-CB4211, and
GKT_137831.
[0153] 52. The method of any one of the aspects 31 to 51, wherein
the subject is administered a total daily dose of 1000 mg to 3000
mg of niacin, or niacin equivalent dosing of a niacin analog
thereof.
[0154] The following examples are intended to illustrate but not
limit the disclosure. While they are typical of those that might be
used, other procedures known to those skilled in the art may
alternatively be used.
EXAMPLES
[0155] Human Donor Subjects' Demographics, Medical History, and
Liver Histology: Cryopreserved primary cultures of human hepatic
stellate cells (HSC, passage 0) isolated from the livers of human
subjects without fibrosis or NASH features and patients with
fibrosis with associated NASH features steatosis, inflammation)
were purchased from Samsara Sciences, Inc., San Diego, Calif. (now
available from LifeNet Health). Detailed donor subjects' history
report including donor demographic characteristics, cause of death,
medical history, and liver pathology provided by Samsara Sciences
are summarized in Table 1. In brief, donor control patients
(non-fibrosis subjects) past medical histories did not present any
major metabolic abnormalities commonly associated with hepatic
fibrosis or NASH. Additionally, liver histological assessment
performed in liver samples in these normal donor subjects by a
certified pathologist showed NAFLD activity score of 0, steatosis
grade 0, inflammation score 0, and fibrosis score 0.
TABLE-US-00001 TABLE 1 Human Donor Subjects Demographics, Medical
History, and Liver Histology Gender, Donor Race, Age Weight (kg),
Cause of Liver Pathology ** Subjects (years) BMI Past Medical
History Death NAS* Steatosis Inflammation Fibrosis 1 Female, 63.6,
27.4 Hyperlipidemia ICH/Stroke 0 of 8 0 of 3 0 of 4 0 of 4
Caucasian, 59 2 Male, 72.7, 22.9 None Trauma 0 of 8 0 of 3 0 of 4 0
of 4 African American, 24 3 Female, 87.6, 34.2 Diabetes type II,
CVA/Stroke 2 of 8 1 of 3 1 of 4 2-3 of 4 Hispanic, Pancreatitis,
high 35 cholesterol, fatty liver, history of Thyroid cancer 4 Male,
89.5, 35 Diabetes Type II, Anoxia/CVD 3 of 8 1 of 3 2 of 4 2 of 4
Hispanic, Hypertension, 45 Schizophrenia 5 Female, 127, 48.06
Hypertension, Diabetes Cardiac arrest/ 3 of 8 2 of 3 1 of 4 1-2 of
4 Caucasian, Type II, Depression Anoxia 53 6 Female, 100, 34.5
Diabetes Type II, Kidney Anoxia/ 4 of 8 3 of 3 1 of 4 1-2 of 4
Asian, disease, Hyper-lipidemia, Diabetic 44 Asthma, Arthritis
Ketoacidosis 7 Male, 113, 37.0 CAD/CABG, Cardiac arrest/ 5 of 8 2
of 3 2 of 4 1 of 4 African Hypertension, Anoxia America,
Hypercholesterolemia 66 *NAS = NAFLD Activity Score: ** NASH CRN
Scoring System (Hepatology 41: 1313-1321, 2005)
[0156] For the study, the stellate cells were selected from
patients who had fibrosis and the features of NASH. The alcohol
consumption and remote past history of viral hepatitis was not
known. Human donor patients with fibrosis had past histories of
varied metabolic abnormalities including type II diabetes,
dyslipidemia, hypertension, and coronary artery disease, etc. (see
Table 1). Only one patient had a living diagnosis of fatty liver
disease. Liver pathology assessment by a certified pathologist
revealed varied degree of liver histology in patients with fibrosis
including NAFLD activity scores 2-5, steatosis grades 1-3,
inflammation scores 1-2, and fibrosis scores 1-3 (see Table 1). The
donor subjects ranged in age from 35-66 years, represented both
genders, and varied racial origins, including Caucasian, Hispanic,
African American, and Asian.
[0157] Detailed product information on individual human hepatic
cells including their morphology and phenotypic characteristics
were previously characterized. Briefly, human normal primary
hepatic stellate cells from normal nonfibrotic subject donors were
characterized by expression of glial fibrillary acidic protein
(GFAP) and to a lesser extent Desmin. Stellate cells from donors
with NASH were characterized by activated phenotype with the
expression of smooth muscle cell actin (SMA) and collagen. The
expression of GFAP, Desmin, SMA were assessed in order to
characterize normal and activated stellate cells from control
(nonfibrotic cells) human donor subjects and donor subjects with
fibrosis. The cultures were also checked for any contamination by
fibroblasts or endothelial cells by checking the expression of TE-7
and CD31, respectively.
[0158] Preparation of Hepatic Stellate Cells. HSC were isolated
based on differential centrifugation through a Nycodenz gradient.
In addition to the isolation methods listed above, the stellate
cells were further purified using cell culture conditions specific
for the cell type. Cryopreserved HSCs were grown in DMEM+10% FBS
media containing 1% antibiotic/antimycotic according to the
recommended media and procedures. HSCs at passage 2 were used for
all in vitro studies described below. During the experimental
incubations with niacin, TGF-.beta. or H.sub.2O.sub.2, cells were
incubated in DMEM+0.5% FBS.
[0159] Hepatic stellate cell fibrosis quantification. Cellular
total collagen and collagen type I content, as an index of
fibrosis, were measured by commercially available Sirius Red
Collagen Detection Kit and Human Type I Collagen Detection Kit
(ELISA) respectively from Chondrex, Inc., Redmond, Wash. Cellular
digestion with pepsin, solubilization, and assay procedures were
performed according to the assay protocols provided in the assay
kits. Collagen and Collagen Type I content using Sirius Red and
ELISA kits were measured based on the standard curve. Additionally,
Sirius Red stained cells were also used for photographic images
(magnification 10.times.) for qualitative visual representation of
collagen content in various treatment groups.
[0160] Reactive Oxygen Species (ROS) quantification. ROS
production, as an index of oxidative stress, in stellate cells was
measured using DCFDA fluorescence as described previously. In
brief, cells were incubated with DCFDA (10 .mu.mol/L) for 30 min.
After washing, the fluorescent intensity in the cell lysate was
measured at the excitation and emission wavelength of 488 and 520,
respectively. Additionally, DCFDA stained cells were also used for
photographic images (magnification 10.times.) for representation of
ROS content.
[0161] Assessment of cell viability. Human hepatic cell viability
was assessed by using commercially available PrestoBlue Cell
Viability Reagent kit by Invitrogen, Carlsbad, Calif. PrestoBlue
Reagent kit utilizes a compound that is quickly reduced by
metabolically active cells, providing a quantitative measure of
viability and cytotoxicity. Cell viability assay and quantitation
by measuring fluorescence (at excitation 540-570 nm, emission
580-610 nm) were performed according to the protocol provided in
the assay kit by Invitrogen.
[0162] Statistical Analysis. Data presented are mean.+-.SE of 3
separate experiments for each subject stellate cells. Statistical
significance was calculated by using Student's t test, and a value
of p<0.05 was considered significant.
[0163] Niacin reverses fibrosis in hepatic stellate cells from
human subjects. For these studies, human hepatic stellate cells
from donor patients with varying degree of fibrosis (F1-3) and NASH
(NAFLD activity scores 2-5) and stellate cells from normal
non-fibrosis subjects (NAFLD activity score of 0) were incubated
with pharmacologically relevant concentrations of niacin (0.25 mM
and 0.5 mM) in DMEM+0.5% media for 48 or 96 hours. Cells were
stained with Sirius Red and collagen content quantitated according
to the procedure noted in Sirius Red Collagen Detection Kit.
[0164] In the representative photographic image of collagen content
in stellate cells from non-fibrosis and from fibrotic NASH subjects
(see FIG. 1A), collagen content in stellate cells from NASH patient
(donor 6) was strikingly higher than in stellate cells from normal
non-fibrosis subject (donor 1) which showed very minimal to Sirius
Red stainable collagen content. Table 2 displays quantitative
changes in collagen content at baseline, 48- and 96-hours
incubation with 0.25 mM and 0.50 mM niacin in stellate cells from
individual normal non-fibrosis subjects (donors 1, 2) and from
patients with fibrosis (donors 3-7). FIG. 1B displays the composite
Mean.+-.SE collagen content data from all 5 fibrosis patient
(donors 3-7) showing the ability of niacin to regress pre-existing
fibrosis in stellate cells from patients with fibrosis. Collagen
content in stellate cells from fibrotic patients were strikingly
higher (4-fold) than in stellate cells from control non-fibrosis
subjects (Table 2, FIG. 1A-B). Treatment of hepatic stellate cells
from patients with fibrosis (donor patients 3-7) with niacin (0.25
mM and 0.5 mM) for 48 h or 96 h, respectively, produced a robust
and significant regression of pre-existing fibrosis by an average
of 47.6 and 60.1 percent (at 48 h incubation) and 53.5 and 65.0%
(at 96 h incubation, p<0.001, respectively (Table 2, FIG. 1B).
At both 48 and 96 h incubation, 0.5 mM niacin had significantly
greater regression of fibrosis than 0.25 mM niacin (60.1 vs 47.6%
at 48 h and 65.0 vs 53.5% at 96 h, p<0.03, Table 2, FIG.
1B).
[0165] It is striking to note that niacin caused a significant
regression of pre-existing fibrosis in stellate cells from all 5
donor subjects. However, niacin did not affect the low measurable
collagen content in stellate cells from normal non-fibrosis
subjects with NAFLD activity score of 0 and fibrosis score of 0
(Table 2).
TABLE-US-00002 TABLE 2 Effect of Niacin on collagen content in
hepatic stellate cells from Non-NASH normal Subjects and NASH
patients with fibrosis Incubation time 48 h 96 h Donor Niacin 0
0.25 0.5 0 0.25 0.5 Subject/Patient (mM) Collagen (.mu.g/mL)
Collagen (.mu.g/mL) Normal Non-Nash 1 21.5 .+-. 2.4 20.0 .+-. 0.2
20.1 .+-. 0.1 23.4 .+-. 2.8 21.9 .+-. 1.5 ND 2 20.4 .+-. 0.2 20.6
.+-. 0.1 24.0 .+-. 1.1 25.2 .+-. 0.6 23.9 .+-. 0.1 ND Mean .+-. SE
21.0 .+-. 1.1 20.3 + 0.2 22.1 + 1.0 24.3 + 1.4 22.9 + 0.8 (Patients
1-2) NASH with Fibrosis 3 60.1 .+-. 1.7 32.7 .+-. 2.7 24.6 .+-. 2.4
94.6 .+-. 5 46.4 .+-. 5.1 29.7 .+-. 5.1 4 73.3 .+-. 5.3 42.3 .+-.
2.2 28.6 .+-. 3.2 88.4 .+-. 3.1 31.4 .+-. 2.7 21.5 .+-. 3.2 5 70.4
.+-. 5.0 24.9 .+-. 4.5 24.3 .+-. 2.0 78.4 .+-. 7.0 22.7 .+-. 3.6
18.8 .+-. 4.0 6 96.5 .+-. 5.6 49.2 .+-. 2.0 34.7 .+-. 2.0 98.9 .+-.
7.6 60.7 .+-. 2.4 40.4 .+-. 4.6 7 123.1 .+-. 7.6 77.9 .+-. 6.4 60.2
.+-. 6.8 130.3 .+-. 7.0 74.3 .+-. 7.2 70.8 .+-. 8.8 Mean .+-. SE
84.7 .+-. 11.3 45.4 .+-. 9.1* 34.5 .+-. 6.7* 98.1 .+-. 8.7 47.1
.+-. 9.3* 36.2 .+-. 9.4* (Patients 3-7) Mean % Regression 47.6 .+-.
4.6 60.1 .+-. 2.5 .sup.a 53.5 .+-. 6.2 65.0 .+-. 5.7.sup.a
(Patients 3-7) *P < 0.001 vs 0 mM Niacin for respective
incubation period .sup.a P < 0.03 vs 0.25 mM Niacin for
respective incubation period; ND, Not determined
[0166] Niacin prevents TGF-.beta. or H.sub.2O.sub.2-induced
fibrosis. Determining whether niacin prevents and reverses stellate
cell fibrosis induced by major physiological stimulators of liver
fibrosis such as TGF-.beta. or oxidative stress mediator hydrogen
peroxide (H.sub.2O.sub.2) was next examined using human hepatic
stellate cells from normal non-fibrosis human donor subjects. For
these studies, human hepatic stellate cells from non-fibrotic
subjects were incubated with TGF-.beta. (20 ng/mL) or
H.sub.2O.sub.2 (25 .mu.M) in the absence or presence of niacin (0.5
mM) for 24 h. Hepatic stellate cell fibrosis was assessed by
measuring collagen content by Sirius Red staining kit. As shown in
the representative photographic images of stellate cells after
staining with Sirius Red (FIG. 2, Top panel), incubation of cells
with either TGF-.beta. or H.sub.2O.sub.2, known inducers of
stellate cell fibrosis, markedly increased collagen content as
compared to vehicle treatment (VEH). Co-incubation of these cells
with either TGF-.beta. or H.sub.2O.sub.2 and niacin for 24 h
noticeably prevented collagen production as shown in cellular
photographic images after Sirius Red staining (FIG. 2, Top panel).
Treatment of stellate cells from normal non-NASH subjects with
niacin (0.5 mM) in the absence of TGF-.beta. or H.sub.2O.sub.2 had
no effect on collagen content as shown in photographic image
labeled as NIA (FIG. 2, Top panel).
[0167] Quantitative data in FIG. 2, bottom bar diagram showed that
both H.sub.2O.sub.2 and TGF-.beta. robustly and significantly
increased collagen content by 266% and 233% respectively in human
stellate cells from non-fibrosis patients. Treatment of these cells
with niacin in the presence of either H.sub.2O.sub.2 or TGF-.beta.
markedly and significantly prevented collagen production by 59% and
56% respectively when compared to cells treated with H.sub.2O.sub.2
or TGF-.beta. alone (see FIG. 2, bottom bar diagram). Niacin (0.5
mM) in the absence of H.sub.2O.sub.2 or TGF-.beta. had no effect on
cellular collagen content (see FIG. 2 bottom bar diagram, last
column labeled as NIA 0.5 mM).
[0168] Niacin reverses TGF-.beta. or H.sub.2O.sub.2-induced hepatic
stellate cell fibrosis. In these studies, human hepatic stellate
cells from non-fibrosis subjects were first stimulated with
TGF-.beta. (20 ng/mL) or H.sub.2O.sub.2 (25 .mu.M) for 24 h to
induce fibrosis. These cells were then continued to incubate
additional 24 h in the absence or presence of niacin (0.5 mM).
Cellular content of Collagen type I was measured by ELISA as noted
above. As shown in FIG. 3, both H.sub.2O.sub.2 and TGF-.beta.
robustly increased by 4-5-fold cellular collagen type I content as
compared to vehicle (control). Treatment of these cells with
pre-existing fibrosis (induced by H.sub.2O.sub.2 or TGF-.beta.)
with niacin (0.5 mM) almost completely reversed cellular fibrosis,
and collagen type I contents were like the controls (see FIG. 3).
Like FIG. 2 data, treatment of cells with niacin (labeled as Niacin
0.5 mM) without previous stimulation with H.sub.2O.sub.2 or
TGF-.beta. had no effect on collagen type I content (see FIG. 3,
last column).
[0169] Niacin prevents human hepatic stellate cell oxidative stress
induced by palmitic acid or H.sub.2O.sub.2. Since oxidative stress
with increased cellular ROS plays an important role in hepatic
stellate cell fibrosis, the effect of niacin on ROS induced by
physiological mediators of oxidative stress such as palmitic acid
and H.sub.2O.sub.2 was next investigated. For these studies, human
hepatic stellate cells from normal non-fibrosis subjects were first
stimulated with either palmitic acid (0.5 mM) or H.sub.2O.sub.2 (25
.mu.M) for 24 h to induce oxidative stress. These cells stimulated
with palmitic acid or H.sub.2O.sub.2 were then continued to
incubate additional 24 h in the absence or presence of niacin (0.5
mM). Representative cellular photographic images after staining
with DCFDA (see FIG. 4 upper panel) showed that both palmitic acid
(PA) and H.sub.2O.sub.2 dramatically induced stellate cell ROS
levels by 3.6- and 5.2-fold respectively, compared to vehicle (VEH)
treatment (quantitatively shown in FIG. 4 lower panel). Treatment
of these cells with pre-existing oxidative stress (induced by
palmitic acid or H.sub.2O.sub.2) with niacin (0.5 mM) significantly
and visibly decreased cellular ROS levels by 52% and 50%
respectively (see FIG. 4 lower panel). Treatment of cells with
niacin (labeled as NIA) without previous stimulation with palmitic
acid or H.sub.2O.sub.2 had no effect on baseline low ROS
levels.
[0170] Niacin did not affect human hepatic stellate cell viability:
For these studies, human hepatic stellate cells were incubated with
niacin (0-0.5 mM) for 48 h or 96 h. Cellular viability was measured
by a cell viability assay kit as noted above. As shown in FIG. 5,
treatment of cells with niacin (0.25 mM or 0.5 mM) for 48 h and 96
h had no significant effect on cell viability as compared to
stellate cells without incubation with niacin.
[0171] A number of embodiments have been described herein.
Nevertheless, it will be understood that various modifications may
be made without departing from the spirit and scope of this
disclosure. Accordingly, other embodiments are within the scope of
the following claims.
* * * * *